A Comprehensive Biological and Clinical Perspective Can Drive a Patient-Tailored Approach to Multiple Myeloma: Bridging the Gaps between the Plasma Cell and the Neoplastic Niche

Antonio Giovanni Solimando; Angelo Vacca; Domenico Ribatti

doi:10.1155/2020/6820241

A Comprehensive Biological and Clinical Perspective Can Drive a Patient-Tailored Approach to Multiple Myeloma: Bridging the Gaps between the Plasma Cell and the Neoplastic Niche

Solimando, Antonio Giovanni;Vacca, Angelo;Ribatti, Domenico 2020-05-18 00:00:00 Hindawi Journal of Oncology Volume 2020, Article ID 6820241, 16 pages https://doi.org/10.1155/2020/6820241 Review Article A Comprehensive Biological and Clinical Perspective Can Drive a Patient-Tailored Approach to Multiple Myeloma: Bridging the Gaps between the Plasma Cell and the Neoplastic Niche 1 1 2 Antonio Giovanni Solimando, Angelo Vacca, and Domenico Ribatti Department of Biomedical Sciences and Human Oncology, Section of Internal Medicine “Guido Baccelli” University of Bari Aldo Moro, Piazza Giulio Cesare, 11, 70124 Bari, Italy Department of Basic Medical Sciences, Neurosciences and Sensory Organs, University of Bari Medical School, Bari, Italy Correspondence should be addressed to Domenico Ribatti; domenico.ribatti@uniba.it Received 21 December 2019; Revised 17 February 2020; Accepted 10 April 2020; Published 18 May 2020 Guest Editor: Klaus Podar Copyright © 2020 Antonio Giovanni Solimando et al. (is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (ere is a broad spectrum of diseases labeled as multiple myeloma (MM). (is is due not only to the composite prognostic risk factors leading to diﬀerent clinical outcomes and responses to treatments but also to the composite tumor microenvironment that is involved in a vicious cycle with the MM plasma cells. New therapeutic strategies have improved MM patients’ chances of survival. Nevertheless, certain patients’ subgroups have a particularly unfavorable prognosis. Biological stratiﬁcation can be subdivided into patient, disease, or therapy-related factors. Alternatively, the biological signature of aggressive disease and dismal therapeutic response can promote a dynamic, comprehensive strategic approach, better tailoring the clinical management of high- risk proﬁles and refractoriness to therapy and taking into account the role played by the MM milieu. By means of an extensive literature search, we have reviewed the state-of-the-art pathophysiological insights obtained from translational investigations of the MM-bone marrow microenvironment. A good knowledge of the MM niche pathophysiological dissection is crucial to tailor personalized approaches in a bench-bedside fashion. (e discussion in this review pinpoints two main aspects that appear fundamental in order to gain novel and deﬁnitive results from the biology of MM. A systematic knowledge of the plasma cell disorder, along with greater eﬀorts to face the unmet needs present in MM evolution, promises to open a new therapeutic window looking out onto the plethora of scientiﬁc evidence about the myeloma and the bystander cells. urine light chains. (ese parameters may be useful to foresee 1. Introduction the patient characteristics from a biological standpoint, in Multiple myeloma (MM) is an incurable haematological order to predict therapy response and estimate the MM load malignancy characterized by a clonal proliferation of plasma [3]. Nonetheless, the D&S is aﬀected by observer-related bias cells that accumulate preferentially in the bone marrow in quantifying lytic lesions, and since 2005, it has been (BM). It accounts for 1% of all cancers and 10% of all replaced by the International Staging System (ISS), which is haematological malignancies. Resistance to chemotherapy based only on the combination of two parameters, namely, poses one of the main challenges in MM management [1]. β2-microglobulin and albumin [4]. Nowadays, sensitivity Indeed, although advances in MM pathophysiological and speciﬁcity of bone disease identiﬁcation have improved, deconvolution and therapeutic knowledge, MM is still an thanks to the widespread use of computed tomography and incurable disease [2]. According to Durie–Salmon (D&S) of functional imaging such as magnetic resonance and clinical staging, MM patients can be stratiﬁed based on positron-emission tomography (PET scan) [1]. Moreover, available clinical parameters, such as haemoglobin, serum among the three stages, data on ISS stage III, associated with calcium value, X-ray bone study, immunoglobulins, and the poorest outcome, are now available. Since cytogenetics, 2 Journal of Oncology evaluated by ﬂuorescent in situ hybridization (FISH), is Current studies are focused on the bone marrow mi- also a major prognostic factor, a new paradigm shift in croenvironment and inﬂammatory cells as attractive drug- gable targets [15]. (e MM physiology oﬀers a wide range of patient risk stratiﬁcation has incorporated the three re- current genetic abnormalities, the t(4; 14), deletion(17p), targeting opportunities, which can be useful in chemo- and t(14; 16), that are mostly associated with a poorer therapeutics for devising more personalized therapy for MM outcome. (ese are used, along with the clinical and patients [16]. For example, increased BM hypoxia is asso- laboratory parameters, in order to gain a more reliable ciated with increased recirculation of MM plasma cells MM risk classiﬁcation according to the revised ISS (R-ISS) (MM-PCs). Oxygen delivery decrease, by enhancing hyp- [5]. Undoubtedly, in MM, the genomic landscape has a oxia-inducible factor-2 alpha (HIF-2α) activity, induces strong impact on patient outcome and response to therapy MM-PC chemokine ligand 12 (CXCL12) upregulation, with [6–8]. Nevertheless, the disease aggressiveness is not only a diminished migration toward CXCL12 and reduced ad- linked to multistep genetic events but also to the MM hesion to mesenchymal stromal cells in vitro. HIF-2α also microenvironment and the MM bystander cells, involved strongly induced the expression of chemokine receptor 1 (CCR1) in MM-PCs. CCR1 enhances MM-PC dissemina- within the tumor niche in a vicious cycle that leads to MM evolution into more complex pathological architecture tion toward CCL3, while decreasing the MM-PC motility [9]. BM microenvironment-mediated drug resistance is reaction to CXCL12. Additionally, CCR1 upregulation by the main mechanism allowing MM to evade the eﬀects of MM-PCs was correlated with a poor outcome in newly conventional and new drugs [10]. To date, a plethora of diagnosed MM subjects and associated with enhanced cir- pathophysiological mechanisms has been dissected, but culating MM-PCs in these individuals. Taken together, these potential targets considered suitable for therapeutic in- data suggest a role for hypoxia-mediated CCR1 upregulation terventions aimed at interfering with the mutual inter- in driving the egress of MM-PCs from the BM. Targeting actions between the clonal plasma cells and the tumor CCR1 may be a novel strategy to prevent dissemination and milieu have proven largely unsatisfactory [11]. In this overt relapse in MM [17]. scenario, we have carried out an extensive literature re- Mesenchymal stem cells (MSCs), one of the main cell components within the BM milieu, can disseminate toward view to probe the novel insights available from transla- tional investigations, in order to reach a deeper primary tumors and metastatic sites, implying that these understanding of the emerging therapeutic window from cells might modulate tumor growth and metastasis [13]. a bench-to-bedside standpoint. Myeloma-derived MSCs can deeply impact the disease homeostasis. (erefore, MSCs do not represent bystanders in the BM niche but rather dynamic actors in the MM 2. The Role of the Bone Marrow biology. MSCs can represent a novel target to develop the Microenvironment: Novel Molecular next generation of therapy in cancer, both by in vitro en- gineering as antitumor carrier to the tumor sites. MM is no Dependencies in Multiple Myeloma exception to this principle [18]. MSCs were lentivirally Signals from the bone marrow microenvironment play a engineered with osteoprotegerin (OPG) in preclinical pivotal role in supporting MM cell growth, spread, and models aimed to halt MM-related skeletal lesions [19]. (e survival, as well as MM progression [12]. In the bone ﬁrst-in-class proteasome inhibitor bortezomib shapes the marrow microenvironment, the cellular compartment tumor-friendly MM environment by inducing bone matrix consists of hematologic and nonhematopoietic cells such as remodelling [20] and by interfering with MSC diﬀerentia- stromal cells, ﬁbroblasts, osteoblasts, osteoclasts, endothelial tion toward the osteoblastic phenotype [21]. (erefore, cells (ECs), B cells, T cells, natural killer (NK) cells, mac- combination strategies combined proteasome inhibition rophages, mast cells, and myeloid-derived suppressor cells with both vitamin D [22] and epigenetic regulators [23]. (MDSCs). During MM development, MM cells can aﬀect the Building on these strategies, diﬀerent groups unravelled BM cells through cell-cell contact or the secretion of soluble novel mechanisms able to mobilize and eradicate niche- factors to build up a favorable microenvironment. MM cells protected myeloma cells by employing histone deacetylase adhere to bone marrow stromal cells (BMSCs) and trigger inhibitors (HDACis) [24]. Pharmacological interfering with many pathways in the latter, resulting in the transcription nucleosome conformation changes and skeletal metabolism and secretion of multiple cytokines such as interleukin-6 demonstrated the interruption of the molecular crosstalk (IL-6), insulin-like growth factor-1 (ILGF-1), vascular en- between MM cells and the stroma and uncovered indirect dothelial growth factor (VEGF), and stromal cell-derived eﬀects halting cell proliferation, bone disease, and angio- factor-1α (SDF-1α) which mediate MM cell growth, pro- genesis, in vitro and in vivo [24–26]. liferation, survival, and drug resistance [13]. Next, MM cells (e myeloma microenvironment is also characterized by educate the bone marrow cells to support neoplastic cell Notch signalling hyperactivation due to the increased ex- growth, survival, and the acquisition of drug resistance pression of Notch 1 and 2 and the ligands Jagged 1 and 2 in resulting in disease relapse, conferring a survival advantage. tumor cells. Notch activation inﬂuences myeloma cell bi- (e tumor microenvironment is recognized as one of the ology and promotes the reprogramming of bone marrow leading factors promoting chemoresistance, but the mech- stromal cells. Colombo et al. [27] uncovered Jagged blocking anisms responsible for this eﬀect are still largely obscure to be relevant for dismal sensitivity to alkylating agents, [14]. immunomodulatory drugs (IMiDs), and proteasomal Journal of Oncology 3 block MM homing and enhance drug eﬃcacy as well. In inhibition due to MM cell and tumor milieu-related mechanisms. Enhanced CXCR4/SDF-1 alpha signalling is frame of this thinking, Natoni et al. [30] have shown that BMSCs can nurse MM by shaping an immune-tolerogenic boosted by Notch overactivation within the MM environ- ment. Additionally, this chemokine axis fuels antiapoptotic milieu and uncovered sialylation as an actionable mecha- mechanisms [27], prompting therapeutic approaches nism to boost the immune response [30]. holding the potential to interrupt the vicious cycle between the tumoral PCs and the BMSCs and, conceivably, improve 2.1. Angiogenesis in Multiple Myeloma. In 1994, Vacca et al. patients’ responses to standard-of-care therapies [27]. Furthermore, CXCR4/SDF-1 alpha signalling has been [37] demonstrated for the ﬁrst time that bone marrow microvascular density (MVD) was signiﬁcantly increased in revealed to impact clinical outcome in PC dyscrasias. MM compared to monoclonal gammopathies of undeter- Nevertheless, treatment strategies pinpointing this receptor mined signiﬁcance (MGUS) and even more in active vs. or its cognate ligand (burixafor or plerixafor) deemed not nonactive forms. (e close association between angiogenesis adequately proﬁcient. (erefore, a deeper characterization and active MM indicates that it is the vascular phase of of the biological CXCL12/CXCR4 interaction can oﬀer plasma cell tumors. Conversely, MGUS and nonactive MM additional insights, overcoming PC disorder treatment re- sistance and clonal resilience. (is could allow envisioning a represent the avascular phase. (e microvessel area and the labeling index (LI) percent are closely associated with the novel therapeutic window and a more eﬀective drug com- bination, designed to halt myeloma progression [28, 29]. MM activity phase and are mutually correlated [38–41]. In 2011, Ria and colleagues [15] also highlighted neo- Additionally, glycosylation, by modulating diﬀerent aspects of tumor biology, can be considered as a hallmark of vascularization as a constant hallmark of MM progression. (is process is only partially supported by factors such as cancer. Several solid and haematological malignancies are VEGF, ﬁbroblast growth factor-2 (FGF-2), and metal- characterized by enhanced sialylated glycan expression, with loproteinases (MMPs), which are directly secreted by the a direct correlation with higher disseminated behaviour. tumor cells. As a consequence of plasma cell-stromal cell Sialylation can also prime MM homing into its environment interactions, the cytokines within the MM niche, in par- by physical interaction between skeletal precursors, stromal ticular IL-6, drive the release of angiogenic factors from cells, and MM cells creating niches and educating bone cells. (erefore, interfering with sialylation may promote trans- bystanders in the bone milieu, these being one of the main triggers of the angiogenic switch during disease progression. lational navigation of the milieu-drug resistance boundaries and deﬁne alternative combinatorial treatment strategies But along with angiogenesis, vasculogenesis also occurs in the tumor niche of MM subjects, enhancing the vascular tree bringing sialylation inhibitors to the MM-stroma interface formation. In the neoplastic microenvironment of MM [30]. (us, nanotechnologically engineered tools provided individuals, hematopoietic stem cells are primed to become next-generation strategies for tailored anti-MM therapy by ECs by the angiogenic cytokines shed in autocrine, para- optimization of pharmacokinetics and pharmacodynamics crine, and endocrine fashion. proﬁle of conventional chemotherapeutic agents [31]. In (erapeutic strategies in MM consist of conventional detail, novel anthracycline preparation integrating integrin α4β1 within nanoparticles seems to be able to exert enhanced chemotherapy and biologically based therapy targeting not only MM-PCs but also the microenvironment and angio- anti-MM and dismal oﬀ-target eﬀects, oﬀering a proof of concept of the value of this pharmacokinetics innovation genesis. Bortezomib regulates many cellular processes, in- cluding the modulation of transcription factors, such as NF- [31]. An alternative approach was delivering liposomal formulation carrying combinations of taxanes, alendronate, κB, cell cycle progression, inﬂammation, immune surveil- lance, growth arrest, and apoptosis. NF-κB is a major and isoform-adapted transferrin, enabling microenviron- transcriptional factor which mediates the expression of ment drug modulation [32]. Notably, novel engineered many proteins including cytokines, chemokines, and cell tagging strategies combined modern immunotherapeutic adhesion molecules. Bortezomib inhibits NF-κB, enhancing targeting with either proteasome inhibitor [33] or bone- the susceptibility of MM plasma cells to therapeutics, while modifying agents (BMAs) [34], gaining more eﬃcient oﬀ- the induction of IL-6 by BMSCs mediated by NF-κB in- target proﬁle. Speciﬁcally, CD38 receptor- and B-cell mat- uration antigen- (BCMA-) directed approaches have in- creases the secretion of VEGF from MM-PCs. Furthermore, bortezomib inhibits MMEC mitotic activity, through inhi- troduced a practice change in immunotherapy and are being intensively investigated in MM [34, 35], since these mole- bition of VEGF, IL-6, insulin-like growth factor-1 (IGF-1), and angiopoietin-1 and angiopoietin-2 (Ang-1 and Ang-2) cules are highly expressed on the malignant plasma cells. Sialylation inhibition using these approaches also [42]. (e circulating levels of Ang-1, Ang-2, VEGF, and promises incremental activity in interfering within the MM- angiogenin were measured in 54 patients with smouldering niche vicious cycle. Recently, sialyltransferase inhibitors MM (SMM). (is result was compared with those of 27 restored aﬀective anti-MM activity by restoring innate and MGUS patients, 55 MM patients, and 22 healthy controls, acquired immune response, while halting malignant cell demonstrating that the ratio of circulating Ang-1/Ang-2 was proliferation at the same time [36]. reduced in MM individuals with full-blown overt MM due to Currently, delivery systems employing the sialylation inhibitor 3Fax-Neu5Ac encapsulation in combination with a biologically signiﬁcant enhancement of Ang-2, but not in SMM or MGUS nonmalignant control subjects. VEGF and BMA are intensively investigated, in order to potentially 4 Journal of Oncology angiogenin were increased in all patients compared to Bisphosphonates exert a direct eﬀect on MM plasma controls. However, circulating VEGF was higher in symp- cells [55]. In detail, both zoledronic acid and neridronate tomatic MM compared to SMM and MGUS, while angio- have a cytotoxic activity on tumor cells and inhibit an- genin was reduced. Hence, the above data show that the giogenesis [55–57]. (e side eﬀect is osteonecrosis of the Ang-1/Ang-2/Tie-2 axis may be an eﬀective target for the jaw (ONJ), a long-lasting disorder that occurs mainly in development of novel antimyeloma agents [43]. Bortezomib breast cancer and MM patients treated with intravenous downregulates not only the caveolin-1 tyrosine phosphor- bisphosphonates [58]. ylation, responsible for VEGF-mediated MM cell migration, Recently, the role of CX3CL1/fractalkine has been but also the caveolin-1 phosphorylation induced by VEGF in reported, as a novel mechanism of this cell signalling ECs. Finally, bortezomib inhibits the transcription of ICAM- boosting angiogenesis and inﬂammation in multiple 1, VCAM1, and E-selectin [44]. myeloma. ADAM10 and ADAM17 are responsible for (alidomide is an antiangiogenic drug. It modulates tu- cleavage and shedding, thereby modulating CX3CL1/ mor necrosis factor-alpha (TNF-α) signalling through direct fractalkine release. Notably, these MMPs are regulated by and indirect eﬀects on the tumor microenvironment [45]. It inﬂammatory and angiogenesis processes. Firstly, as- also reduces FGF-2 [46], VEGF, and IL-6 secretion by BMSCs sessment of soluble levels in plasma cell disorders at and MM cells [47], stimulating the activation and expansion of diﬀerent disease stages demonstrated that circulating T cells and enhancing NK-cell-mediated cytotoxicity. (a- concentration of CX3CL1 was signiﬁcantly higher in full- lidomide disrupts the host marrow-MM cell interactions by blown disease compared with controls [59]. Strikingly, selectively modulating the density of cell surface adhesion this observation was correlated with BM microvessel molecules. Nonetheless, treatment with thalidomide induces density. Next, ensuing functional in vitro experiments side eﬀects while lenalidomide and pomalidomide, its deriv- recapitulated fractalkine dynamics, highlighting the atives, are both less toxic and more potent [48, 49]. Cereblon theragnostic role of enhanced production of this che- (CRBN) is a primary target of thalidomide teratogenicity, but mokine by MM-derived BM endothelium upon exoge- it is also required for the antimyeloma activity of thalidomide nous stimulus [59, 60]. In fact, Tanaka et al. [61] uncovered mAb-blocking strategies anti-CX3CL1 as next- and related drugs (IMiDs). A decreased CRBN expression is linked to pharmacological resistance in human MM cell line generation approach aimed at halting innate and adaptive models and primary cells and may also provide a biomarker to immune-dependent inﬂammation. Finally, Chen et al. predict IMiD response and resistance [49]. In fact, other also corroborated this evidence demonstrated decreased authors analysed the inﬂuence of the single-nucleotide poly- CX3CL1 production in vivo, upon proteasome inhibition morphisms (SNPs) of the CRBN gene on the risk of adverse [62]. (ese compelling data envision also the use of anti- eﬀects of thalidomide-based chemotherapy in patients with TNF-α, in combination with the abovementioned thera- MM [50]. peutic strategies for MM patients [59]. However, Curry and colleagues found no reduction in MVD before or after treatment with thalidomide of newly diagnosed MM (NDMM) patients [51]. Nevertheless, other 2.2. Endothelial Cells. MMECs express VEGF/VEGFR-2, authors showed that high MVD at diagnosis was considered FGF-2/FGF-2 receptor-2 (FGF-2R-2), and Ang-2/Tie-2 and exert an increased in vitro and in vivo angiogenic activity an independent poor prognosis factor [52]. Lamanuzzi and colleagues [53] evaluated mTOR acti- [63]. Moreover, MMECs express CXC chemokines CXCL8/ IL-8, CXCL11/interferon-inducible T-cell alpha chemo- vation in ECs from 20 patients with MGUS and 47 patients with MM and its involvement in angiogenesis. mTOR and attractant (I-TAC), CXCL12/SDF-1α, and CCL2/monocyte chemotactic protein-1 (MCP-1), which mediate plasma cell the rapamycin-insensitive companion of mammalian target of rapamycin (RICTOR), two components of mTORC2 proliferation and homing [64]. Long-term MMECs were compared with MGUSECs complex [54], were deemed signiﬁcantly elevated in MMECs compared to MGUS-ECs. (e authors uncovered mTORC2 and HUVECs as their normal quiescent counterpart [63]. MMECs but not healthy cells overexpressed endothelial expressed by MMECs to be relevant for angiogenic boosting activation markers [65]. Mechanistically, MMECs in- and found that mTOR/RICTOR targeting by siRNA and dual mTOR inhibitor PP242 reduced the MMEC angiogenic creasingly produce FGF-2, VEGF, MMP-2, and MMP-9 compared to HUVECs, conferring a growth advantage functions, including cell migration, chemotaxis, adhesion, invasion, in vitro angiogenesis on Matrigel over controls by a faster establishment of a proangio- , and cyto- genic phenotypic behavior, in terms of capillary skeleton reorganization. Additionally, in the chick embryo chorioallantoic membrane (CAM) and in Matrigel plug sprouting and net formation [65]. MMECs also boost a strong proangiogenic response in the CAM [66]. Gene assays, PP242-directed approaches demonstrated angiogenic blockade in vivo by interfering with angiogenesis. PP242 expression assays corroborated these pieces of evidence, uncovering MMECs’ phenotype to be characterized by exerted a synergistic eﬀect with IMiDs and proteasome inhibitor, suggesting that mTOR blockade can enhance the enhanced proangiogenic gene transcription, namely, VEGF, FGF isoforms, HGF, Tie2, transforming growth antiangiogenic eﬀect of these drugs. Because mTORC2 in- factor-beta (TGF-β), GRO-α chemokine, ﬁbronectin-1, volved in MM angiogenesis, dual mTOR inhibitor PP242 HIF-1α, ETS-1, ID3, and osteopontin compared to could support antiangiogenic management of MM patients HUVECs. [53]. Journal of Oncology 5 MMECs alone displayed a VEGF-dependent autocrine 2.3. Macrophages and Mast Cells. Tumor-associated mac- growth loop [65], owing to high VEGF and VEGFR-2 ex- rophages and mast cells support tumor growth and neo- vascularization by producing a wide array of angiogenic pression, constitutive autophosphorylation in both VEGFR- 2 and the associated kinase ERK-2, along with the inhibition cytokines. Mast cell- and macrophage-derived growth fac- of proliferation, capillarogenesis, and phosphorylation by tors that can promote tumor development and angiogenesis neutralizing anti-VEGF and anti-VEGFR-2 antibodies. include TNF-α, TGF-β1, FGF-2, VEGF, platelet-derived Pentraxin 3 aﬀected MMEC functional activities and was growth factor (PDGF), IL-8, osteopontin, and nerve growth able to modify the angiogenic capability of both MMECs and factor (NGF). Conversely, mast cell- and macrophage- plasma cells [67]. produced cytokines that may participate in antitumor re- Comparative gene expression proﬁling was made of sponses include IL-1, IL-2, IL-4, IL-10, and interferon- MMECs and MGUSECs with Aﬀymetrix U133A arrays gamma (IFN-c) [73]. [68]. Expression of 22 genes deemed signiﬁcantly diﬀerent When BM macrophages from MM patients are exposed by comparing MMECs with MGUSECs. Key biological to VEGF and FGF-2, they transform into cells that are functionally and phenotypically similar to paired MMECs, processes related to protumorigenic functions were af- fected, showing signiﬁcant gene expression deregulation and generate capillary-like networks mimicking those of in the symptomatic disease when compared to the pre- MMECs [74]. Macrophages from nonactive MM, MGUS, cursor’s states. Next, DIRAS3, SERPINF1, SRPX, BNIP3, and benign anaemia patients display similar albeit weaker IER3, and SEPW1, gene-encoding proteins, were func- features. EC-like macrophages and apparently typical tionally tested to substantiate the gene signature ﬁndings, macrophages contribute considerably to the formation of corroborating their proangiogenic function in BMECs. new vessels in patients with active MM, whereas their BNIP3, IER3, and SEPW1 transient gene silencing had a vascular supply is minimal in nonactive MM and absent in signiﬁcant impact on programmed death, cell prolifera- MGUS patients and control patients [74]. In contrast to tion, adhesion, and angiogenesis-related functions. Four MGUS and asymptomatic disease, CD14/CD68 surface proteins were found to be overexpressed in MMECs: overexpression has been found in full-blown myeloma. BM trephine immune staining additionally dissected two mac- ﬁlamin A, vimentin, α-crystallin B, and 14-3-3ζ/δ protein [69]. (eir expression was enhanced by VEGF, FGF-2, rophage subpopulation, demonstrating cells with either HGF, and MM-PC-conditioned medium and their si- endothelial or conventional phenotype by CD68/FVIII-RA lencing RNA knockdown aﬀected MMEC angiogenesis- coloration. Remarkably, proangiogenic eﬀects on macro- related functions, including spreading, migration, and phages have shown to contribute to the building of neovessel tubular morphogenesis [69]. wall in patients with active MM over nonactive and MGUS More recently, Leone described a novel aspect of conditions [74]. disease pathophysiology, by characterizing the MM cell Proinﬂammatory macrophages in BM biopsies are a interface with the local environment, namely, vascular potential prognostic biomarker for acquired MM resistance endothelium between ECs and CD8 lymphocyte, create a to bortezomib therapy, and high levels in BM are correlated permissive immune microenvironment within the BM, with poor survival. Remarkably, proteasome inhibitor treatment of proinﬂammatory macrophages primed MM- allowing plasma cell proliferation. In this context, the corrupted endothelium behaves as tolerogenic promoter, tumor-initiating cell (MM-TIC) inﬁltration both in vitro and in vivo in an IL-1β-dependent fashion. One way to abolish by indirect negative regulation of the eﬀector memory + + CD8 T cells. (e CD8 T-cell population sustained by bortezomib-induced MM-TIC enrichment is by blocking ECs also expressed Foxp3, producing IL-10 and TGF-β, the IL1β axis using a pharmacologic or genetic approach and exerting a protumorigenic activity. (e above study [75]. Additionally, CD163 expression was detected by im- adds further insight into the role that ECs play in MM munohistochemistry to determine the number of tumor- biology and describes an additional immune regulatory associated macrophages (TAMs) in 198 MM patients re- mechanism that inhibits the development of antitumor ceiving bortezomib-based regimens. Enhanced CD163+ TAM inﬁltration in NDMM was correlated with a worse immunity and may impair the success of cancer immu- notherapy [70]. clinical outcome, in terms of progression-free survival (PFS), overall survival (OS), and a worse therapy response quality Extracellular vesicles (EVs) shed from the MM cell surface actively participate in cellular crosstalk and vessel compared with subjects with lower CD163+ TAM inﬁltra- tion. (ese data indicate that the CD163+ TAM content at formation during MM progression [71]. Proteasome inhi- bition via Bi-EVs decreased EC proteasome activity, and Bi- diagnosis is a powerful predictor of prognosis in MM [76]. EVs released from apoptotic MMECs promoted angiogen- Another link between the eﬀect of bortezomib in MM pa- esis suppression by decreasing the proliferation and mi- tients and macrophages was highlighted by Khalife’s studies. gration of ECs [71] IMiDs exerted a relevant antiangiogenic (ey demonstrated the improved treatment eﬀectiveness eﬀect in vivo, and in vitro, it also inhibited migration of gained by miR-16 increased expression in boosting anti-MM MMECs, but not of MGECs or control HUVECs. VEGF/ activity by a proteasome inhibitor in the presence of MM resident TAMs [77]. Enhanced soluble miR-16 in MM in- VEGFR-2 cell signalling was deemed biologically connected to lenalidomide treatment, which exerted a signiﬁcant im- dividuals linked to more favourable outcome. Conversely, deletion 13 on cancer cells was inversely associated with pact on cytoskeleton rearrangement, migration, and cell metabolism in MMECs [72]. peripheral miR-16 concentration [78]. miR-16 can be 6 Journal of Oncology Additionally, urinary N-terminal propeptide of procollagen actively secreted by MM cells through EVs, with a direct correlation between intracellular and shed levels. EVs iso- type I (Ntx) concentrations was assessed by an enzyme- linked assay, at diﬀerent disease stages and bone involve- lated from MM patients can drive circulating monocyte diﬀerentiation to M2-TAMs, while the increased concen- ment. Enhanced mast cell count, RANKL, and Ntx con- tration of circulating miR-16 reverts this behaviour. In vivo, centrations were found in MM subjects. Furthermore, mast miR-16 lost sustains macrophage diﬀerentiation toward an cell density was positively correlated with MMP-9, RANKL, M2 phenotype acquisition, most likely due to dismal NF-κB and Ntx. (erefore, mast cells may contribute to osteolytic activation via IKKα/β targeting [77]. Moreover, the immune processes during MM progression [88]. function of macrophages is mediated by IL-32c, which is overexpressed in MM patients and associated with a more 2.4. Cancer-Associated Fibroblasts (CAFs) in MM. In the advanced clinical stage [79]. Gene expression proﬁling progression of the disease from MGUS to MM, a ﬁbroblast showed a signiﬁcant IL-32c-dependent induction of the switch is required to acquire protumorigenic activity and immunosuppressive molecule indoleamine 2,3-dioxygenase parallels the behaviour exhibited by other haematological (IDO) in macrophages, and this eﬀect was veriﬁed by qRT- and solid cancers [89–92]. (e switch was demonstrated by PCR, western blotting, and immunoﬂuorescence. Proteinase the bone marrow ﬁbroblast gene expression proﬁle of pa- 3 (PR3), an IL-32 binding protein, was universally expressed tients with MGUS and MM, extracted by nonnegative matrix on the surfaces of macrophages, and PR3 knockdown or the factorization (NMF) [93]. Moreover, a speciﬁc miR proﬁle in inhibition of the STAT3 and NF-κB pathways hindered the BM ﬁbroblasts is linked to the transition from the asymp- IL-32-gamma-mediated stimulation of IDO expression. tomatic to the full-blown disease. BM ﬁbroblasts and EV- (ese results indicate that MM cell-derived IL-32c promotes dependent vicious cycle orchestrated by MM cells determine the immunosuppressive function of macrophages and is a an enhanced production of miR-27b and miR-214, fuelling potential target for MM treatment [80]. proliferative and antiapoptotic pathways. (ese prosurvival MGUS and smouldering disease seem to be character- functions parallel an increased expression of ﬁbroblast ac- ized by a peak of mast cell density count-related and an- tivation markers alpha-smooth muscle actin (αSMA) and giogenesis enhancement [81]. Ang-1 is a crucial promoter of ﬁbroblast activation protein (FAP). While strengthening the MM cell growth by stimulating angiogenesis. Experimental mechanisms involved in the transition from MGUS and evidence indicates that Ang-1 secreted by primary murine SMM to MM, a peculiar miRNA proﬁle in MM-associated mast cells promotes marked neovascularization in an in vivo ﬁbroblasts, along with the myeloma cells, educates the BM transplantation assay [82]. Primary mast cells accelerate microenvironment by priming the BM ﬁbroblast phenotype tumor growth by established plasmacytoma cell lines, while [94]. In fact, Desantis et al. studied the eﬀect of recombinant Ang-1-neutralizing antibodies signiﬁcantly reduced the human erythropoietin (rHuEPO) on MM ﬁbroblasts in vivo growth of plasmacytomas containing mast cells. Moreover, and in vitro. It had previously been demonstrated that mast cell inﬁltrate parallels proangiogenic cytokine con- rHuEPO regulated angiogenic responses in MM via a direct centration, growth-related oncogene-alpha (GRO-alpha), eﬀect on macrophages and ECs. Likewise, rHuEPO de- and epithelial neutrophil-activating protein-78 (ENA-78). creases the activation marker (αSMA and FAP) expression (e authors also demonstrated that mast cell density was in MGUS and MM; furthermore, proinﬂammatory and correlated with ki-67 PI, suggesting an important partici- proangiogenic cytokines, such as IL-6 and IL-8, VEGF-A, pation of mast cells in MM biology and growth [83]; in this FGF-2, and HGF in MM ﬁbroblasts, signiﬁcantly dimin- context, mast cells would enhance angiogenesis, produce ished. Collectively, rHuEPO halted the MM-associated ﬁ- cytokines with growth eﬀects on myeloma cells, and modify broblast proliferation. Conversely, ﬁbroblast-programmed the BM microenvironment [84]. (erefore, mast cells could cell death enhanced in both MGUS and MM. Overall, these be indicators of the disease activity [85] and valuable targets data pinpoint rHuEPO as a key brake on MM-supporting for therapeutic interventions [83]. In line with this view- ﬁbroblast action [95]. point, mast cells may be a novel target for an adjuvant strategy aimed at halting angiogenesis by interrupting the vicious cycle underlying cytotoxic cytokine production, thus 2.5. Myeloid-Derived Suppressor Cells. MDSCs are myeloid circumventing mast cell-mediated immune suppression cells with a speciﬁc inhibitory activity on the immune re- [86]. sponse, which accumulate in the tumor microenvironment BM specimens from active myeloma over premalignant during tumor development [96] Signiﬁcant accumulations derived trephines analysed by both laser and electron mi- of immunosuppressive MDSCs were observed in the BM of croscopy were characterized by the presence of neovessels patients at early stages of MM and regulated MM growth by lined by granulated mast cells [87]. Otherwise, in MGUS, inhibiting T cells [97]. Moreover, murine MM cells directly mast cells are localized on the abluminal side of neovessels activate BM MDSCs and enhance their immunosuppressive function through soluble factors such as granulocyte-mac- [87]. However, mast cell density has an impact not only on angiogenesis but also on the progression of bone disease in rophage colony-stimulating factor (GM-CSF), promoting the immune escape of MM cells [98]. An increase of bone MM patients. In 52 MM patients, BM mast cell density was measured by immunohistochemical staining for tryptase, marrow MDSCs was also detected in the 5T33 MM mouse and serum levels of MMP-9 and RANKL were measured by a model after inoculation with MM cells [98]. In the BM of solid-phase sandwich enzyme-linked immunosorbent assay. 5T33 MM mice, exosomes derived from MM cells increased Journal of Oncology 7 the number of BM MDSCs in vivo and induced changes in dendritic cells shaping within the tumor niche and opening MDSC subpopulations which are similar to their phenotype, novel therapeutic windows [110–112]. All-trans retinoic acid (ATRA) indeed enhanced MAPK activation with dismal suggesting the involvement of exosomes in the accumulation of MDSCs [99]. reactive oxygen species levels, prompting mature myeloid MDSCs can mediate the suppression of myeloma-spe- lineage fuelling [113, 114]. ciﬁc T-cell responses through the induction of T-cell anergy Despite the existence of a correlation between MDSC and Treg development in the MM microenvironment [100]. pathophysiology and proangiogenic factors, VEGF-blocking Polymorphonuclear (PMN)-MDSCs and neutrophils mAb strategies did not succeed. Furthermore, the likelihood of equivalently sustain MM resistance to alkylators and MDSC-induced reduced sensitivity to the antiangiogenetic doxorubicin, by mediating soluble factor production. Tar- therapy discouraged further attempts in this direction [115]. geting PMN-MDSCs could enhance chemotherapy eﬃcacy Conversely, promising results generated by investigating in MM. It is well accepted that targeting MDSCs in cancer miRNA-based approaches [112, 116] hold the potential to improves the immune response and increases the eﬃcacy of reduce MM disseminated potential and provided the bases to revealMDSC-relatedtargetstoidentify,mobilize,anderadicate immunotherapy. MDSCs play an ancillary role as a suitable target to overcome MM drug resistance, an important niche-protected cells likely able to favor MM progression [117]. ﬁnding in light of recent data suggesting the beneﬁt of Aiming to implement MDSC-dependent immunosup- combined chemo- and immunotherapy treatment protocols pression halting strategies, several attempts have been made [100–102]. Due to the loss of equilibrium in the MM im- to interfere with cyclooxygenase-2 (COX-2), arginase-1 mune landscape, immune checkpoint targeting agents have expression, and inducible nitric oxide synthases and to not shown clinical activity in MM. It is therefore critically decrease reactive oxygen species production and provided important to deal with immunosuppressive mechanisms and undeniable rational for the novel association of anti-in- improve immune responses, especially in advanced MM ﬂammatory compound to the MM therapeutic backbone, in patients. New immunotherapeutic strategies such as im- order to expand the eﬀectiveness of immunotherapy and to munomodulatory drug-intensiﬁed monoclonal antibody decrease the myeloid-derived population in the MM envi- ronment [118]. Remarkably, the antigen-presenting cell treatment, immune checkpoint inhibitors, and chimeric antigen receptor T-cell therapy targeting B-cell maturation (APC) capacity of dendritic cells and ECs [76] can also open antigen have been employed in advanced-stage MM patients a further therapeutic window, since several examples have [103]. An association between high PMN-MDSC levels and recently been published, highlighting the tight crosstalk poor overall survival in MM patients has been validated. between the immune microenvironment gene signature, PMN-MDSCs induced piRNA-823 upregulation, which in vascular cells, and molecular targets, in both haematological turn enhanced DNA methylation, thus stimulating the MM and solid cancers [119, 120]. (ese studies point to the WNT cell clonal evolution. Silencing of piRNA-823 in MM cells pathway as a druggable, theragnostic marker with a plethora reduced the stemness of MSCs maintained by granulocytic of eﬀects on the immune microenvironment in cancer [121]. (G)-MDSCs, resulting in a decreased tumor burden and (is complex scenario fostered an intensive translational angiogenesis in vivo [104]. It has also been demonstrated investigation aimed at improving MM immune equilibrium that the proinﬂammatory cytokine IL-18 is critically in- lost via MDSC targeting. Other noteworthy aspects are volved in MM and its levels are associated with MDSCs. IL- related to the immune microenvironmental landscape and 18-deﬁcient mice were remarkably protected from MM its modulation through ﬂuoropyrimidine, nucleoside ana- progression in a CD8 T-cell-dependent manner. Within the logues, and anthracyclines [122]. Nonetheless, state-of-the- BM milieu, IL-18 stimulates MDSCs, sustaining MM pro- art development [123, 124] holds great potential in cir- gression. High levels of BM plasma IL-18 were associated cumventing myeloid-derived immune suppression by in- with poor survival in MM patients. (e above preclinical terfering with critical signals, such as IL-4 receptor α (IL- studies suggested that IL-18 could be a potential therapeutic 4Rα), thus reducing cell proliferation. Moreover, the binding target in MM [105]. Additionally, the estrogen eﬀect in of the aptamer to its speciﬁc receptor led to MDSC depletion and tumor growth. Peptide enrichment in both M- and hematologic malignancies including MM was studied, and treatment with 17beta-estradiol signiﬁcantly promoted the G-MDSCs by phage-dependent strategies led to the devel- opment of a peptibody, via the fusion of peculiar peptide progression of the disease. However, this eﬀect has not been attributed to a direct eﬀect of estrogen on MM cells but was sequences with the Fc portion of murine IgG2b monoclonal antibodies, demonstrating in vivo activity [124]. considered to be mediated through estrogen-induced al- terations in the tumor microenvironment. In particular, it (e main complexities of the MM microenvironment signiﬁcantly increased the ability of MDSCs to suppress cell network are summarized in Figure 1. T-cell proliferation [106]. Multiple myeloma, MM; vascular endothelial growth Botta et al. [107] pointed out developments in MDSC- factor, VEGF; nerve growth factor, NGF; ﬁbroblast growth directed approaches, by suggesting applications toward factor, FGF; interleukin, IL; tumor necrosis factor-alpha, histone-deacetylase inhibitors in MM and uncovered sig- TNF-α; transforming growth factor-beta 1, TGF-β1; platelet- derived growth factor, PDGF; hypoxia-inducible factor-1 nalling pathways involving MDSCs [108, 109], able to halt inﬂammation, by impairing JAK/STAT downstream. Epi- alpha, HIF-1α; runt-related transcription factor, RUNXs; monocyte chemotactic protein 1, MCP 1; insulin-like growth genetic modulation reduced cell ability of monocytic phe- notype granulocytic shift, by promoting macrophages or factor 1, IGF-1; stromal cell-derived factor 1, SDF-1; MIP1; OPG Osteoclasts activated 8 Journal of Oncology VEGF, FGF-2, ANG1, Angiogenesis MM cells HIF-1α MMP, HGF-SF, Endothelial cells VEGF, NGF, RUNXs TGF-β, TNF-α FGF-2, IL-8 TNF-α, Macrophage TGF-β1 VEGF, FGF-2, MCP1 VEGF, FGF-2, MMP Ostepontin IGF-1, SDF1, MIP1 CXC chemokines PDGF DKK1 HGF sclerostin Mesenchymal cell Mast cell WNT BMP4, TGF-β, bFGF Proteinase MMPs VEGF, HGF, bFGF, IL-1, IL-6, TNF-α, VEGF, HGF, Dissemination PTHrP, MCFs, MIP1α IL-6, TNF-α and spreading of VEGF, TGF-β MM cells TNF-α VEGF, HGF, TGF-β, BAFF IL-6, MMPs, IL-1β, SID1α, TNF-α RANKL RANK Stromal cells, IL-6, IL-3 ,OPG, MIPα ﬁbroblasts SID1α, TNF-α, IL-1β Osteoclast IL-6 progenitor VEGF, TNF-α Immunity deﬁciency Bone remodeling MDSCs Monocyte CD8+T cell NK cell Dendritic cell Figure 1: Soluble factors and adhesion-related interactions between MM cells and bone marrow immune microenvironment: MM-PCs prime the tumoral milieu via a plethora of mechanisms. Immune cell function, by deﬁciency in adaptive and innate response dysfunction, and proinﬂammatory cytokine production drive essential signals for microenvironment colonization and interactions. Moreover, MM-PCs control neoplastic survival and dormancy, modulating the response of the BM microenvironment cells to MM dissemination. Bone disease in MM is a prototypical malignant bone microenvironment pathologic condition. By tackling the knowledge gap on skeletal dissemination and disruption and cell-cell and cell-matrix interaction, the prevention and cure of MM progression may be better understood and targeted by immunomodulation, using combinations of MM-PC-directed agents against novel therapeutic targets. angiopoietin 1, ANG1; metalloproteinases, MMP; hepato- evolution uncovered a reciprocal crosstalk between MM cyte growth factor/scatter factor (HGF/SF), chemokine, cells with the surrounding milieu, and compelling veriﬁ- CXC; Dickkopf, DKK; wingless-type MMTV integration site cation designates that angiogenesis and immunosuppres- family, WNT; bone morphogenetic protein-4, BMP4; sion often fuel a simultaneous vicious cycle [70]. parathyroid hormone-related protein, PTHrP; macrophage Consequently, approaches relating to antiangiogenic im- colony-stimulating factors, MCFs; B-cell activating factor, mune mechanisms seem to hold the promise to tip the BAFF; SID1 transmembrane family, member 1, SID1; re- equilibrium of the MM environment and increase clinical ceptor activator of nuclear factor-kappa-Β ligand, RANKL; beneﬁt. (e ﬁrst-in-class drug thalidomide and its deriv- osteoprotegerin, OPG; natural killer, NK; myeloid-derived ative lenalidomide mirrored the abovementioned knowl- suppressor cells, MDSCs. edge, representing one possible translation from bench-to- bedside eﬀorts [51, 126]. But as stated above, the precise target of lenalidomide is cereblon, since low cereblon levels 3. Therapeutic Windows are associated with drug resistance [127–129]. Undeniable A new goal in haematological malignancies is represented by evidence supports the use of drugs that target the BM a treatment approach targeting not only patients with active microenvironment to prevent the progression of SMM or MM, but also those with SMM. (is shift toward early full-blown MM. Additionally, in a mouse model, the use of intervention [125] with the antiangiogenic agents lenali- an antiangiogenic anti-VEGFR-2 antibody in the early domide and dexamethasone demonstrated prolonged dis- stage delayed tumor progression of MM; nonetheless, ease-free survival and OS in patients with SMM. besides IMiDs, angiogenic-directed strategy did not show eﬀective results in the unselected patient subgroup in patients with MM [130]. In order to achieve a patient- 4. Targeting Angiogenesis and the Immune tailored vasculogenic targeting, stringent patient stratiﬁ- Microenvironment in Multiple Myeloma: cation has been proposed by modulating from the critical Current Challenges step of MM evolution that can be critically dependent on vessel supply, such as smouldering phases [131] or extra- (e knowledge of critical pathways supporting angiogen- medullary dissemination [132–134]. esis and creating immunosuppression during MM Osteoblasts Journal of Oncology 9 Table 1: Compounds in advanced investigation targeting MM cells and the tumor microenvironment. Targets (erapeutic agents ∗ ∗ (1) IMiDs (thalidomide , lenalidomide ) Angiogenesis (2) Anti-VEGF Ab (bevacizumab) (3) Tyrosine kinase inhibitor against VEGF (sunitinib) Hypoxia (1) Evofosfamide investigational hypoxia-activated prodrug (1) CXCR4 inhibitors (plerixafor) Stromal cells (2) Anti-CXCR4 Ab (ulocuplumab) (3) CXCL12 inhibitor (olaptesed pegol) (1) Bisphosphonates (2) Anti-DKK1 Ab Osteoclasts and osteoblast (3) Anti-RANKL Ab (denosumab ) (4) Antisclerostin mAb (1) TGF-β antagonists (sotatercept) (2) TNF-α antagonists (etanercept) Cytokine signalling (3) Anti-TNF-α Ab (inﬂiximab) (4) IL-6 antagonist (siltuximab) (1) Anti-PD-1/PD-L1 Ab (pembrolizumab, nivolumab) (2) Anti-CTLA4 Ab (ipilimumab) (3) CAR-T cells Stimulation of anticancer immunity (4) MILs (5) Vaccines (6) Anti-CD38 Ab (daratumumab , isatuximab) (7) Anti-SLAMF7 Ab (elotuzumab ) Immunomodulatory drugs, IMiDs; vascular endothelial growth factor, VEGF; antibody, Ab; chemokine receptor 4, CCR4; chemokine ligand 12, CXCL12; Dickkopf, DKK; receptor activator of nuclear factor-kappa-Β ligand, RANKL; transforming growth factor-beta 1, TGF-β1; tumor necrosis factor-alpha, TNF- α; interleukin-6, IL-6; programmed cell death 1/programmed cell death ligand 1, PD-1/PD-L1; chimeric antigen receptor-T cells, CAR-T cells; marrow inﬁltrating lymphocytes, MILs; self-ligand receptor of the signalling lymphocytic activation molecule, SLAMF7. FDA and EMA approved. (e next breakthrough of therapeutic strategy design is addition, studies of chimeric antigen receptor-T-cell (CAR-T- targeting the MM ecosystem together with the immune cell) therapy targeting BCMA have revealed very high re- microenvironment. (e altered BM niche sustains the pro- sponse rates in heavily pretreated patients with MM [140, 144]. liferation of MM cells, nursed by physical and soluble re- ciprocal interactions educating both the neoplastic and the As pointed out, CXCL12 and its ligand CXCRA can have immune environmental cells [135]. Identiﬁcation, mobiliza- a pathologic role in diﬀerent stages of MM and patient drug tion, and eradication of this niche-protected dormant and resistance, so disrupting the CXCL12-CXCR4 axis might be often pharmacological insensitive cells have been signiﬁcantly a therapeutic opportunity [39]. Roccaro et al. uncovered improved since several trialsinvolving antibodies have proved CXCL12 and CXCR4 as putative targets to halt MM evo- clinical beneﬁts in MM. Anti-SLAMF7 targeting by elotu- lution and extramedullary dissemination in animal models zumab anti-CD38-directed approaches by daratumumab as a [28, 145], indicating broad potential consequences on ad- single agent or with proteasome inhibitor and IMiDs have hesion-mediated MM dissemination [29, 101, 146] and drug shown far more eﬀectiveness and superior activity than the resistance, as in other solid and haematological malignancies standard of care [136, 137]. (e ﬁrst-in-class SLAMF7 tar- [147–149] and prompt clinical validation [150]. (erapeutic interventions with burixafor or plerixafor (CXCR4 antag- geting molecule stimulates NK cells and macrophages; con- versely, CD38-targeting by daratumumab induces the onists) in MM are not eﬃcient enough [39]. immune system triggering toward Treg reduction and by Bisphosphonates (pamidronate or zoledronic acid) [56] enhancing T-helper and cytotoxic lymphocytes [138]. An- and other BMAs, such as DKK1 inhibitors (Dickkopf WNT other target studied especially in melanoma, lung cancer, and signalling pathway inhibitors), antisclerostin mAb [151], and Hodgkin lymphoma is programmed cell death 1 (PD-1) RANKL inhibitors (denosumab) [152], represent an example [139, 140]. In more detail, the pieces of evidence that PD-1/ of attempts to target the disseminated and localized bone PD-ligand 1 (PD-L1) modulation increases T- and NK cell disease eﬀect due to the spreading of malignant plasma cells. antimyeloma eﬀects prompted the use of immune checkpoint Nonetheless, despite encouraging clinical outcome gained in full-blown disease [153], to date no clinical evidence dem- inhibitors in clinical studies. Nevertheless, the anti-PD-1/PD- L1 mAb as a single agent did not provide suﬃcient results. onstrated a survival beneﬁt by treating the asymptomatic version of myeloma [154]. Lack of inﬁltrating eﬀector cells within the MM milieu can explain the modest eﬃcacy demonstrated by these clinical Studies conducted on MM cell lines have shown that the trials [141]. (erefore, drug combination strategies encour- addition of exogenous IL-6 is essential for obtaining the aged clinical trials (NCT02289222 and NCT02331368), growth of neoplastic cells in vitro. By removing IL-6 from uncovering encouraging medical response [142, 143]. In short-term cultures, MM cells die, demonstrating that this Osteoclasts 10 Journal of Oncology Targeting hypoxia Evofosfamide Targeting cytokine signalling TGF-β antagonists (sotatercept) TNF-α antagonists (etanercept) Hypoxia Anti-TNF-α Ab (inﬂiximab) Targeting angiogenesis Endothelial cells IL-6 antagonists (siltuximab) IMiDs (talidomide, lenalidomide) Anti-VEGF Ab (bevacizumab) Angiogenesis Mast cells sunitinib (tyrosine kinase inhibitor againstVEGF) CD8+T cells Targeting stromal cells activity Stimulation of anticancer immunity Anti-PD1/PDL1 Ab CXCR4 inhibitors (plerixafor) Macrophages (pembrolizumab, nivolumab) Anti-CXCR4 Ab (ulocuplumab) CXCL12 inhibitors(olaptesed Anti-CTLA4 Ab (ipilimumab) CAR-T cells pegol) Stromal cells, MILs ﬁbroblasts Vaccines Dendritic Anti-CD38 Ab (daratumumab) cells Anti-SLAMF7 Ab (elotuzumab) Targeting osteoclasts and osteoblasts NK cells Bisphosphonates Anti-DKK1 Ab Anti-RANKL Ab (denosumab) MDSCs Antisclerostin mAb Figure 2: (erapeutic targets within the tumor milieu in multiple myeloma. Immunomodulatory drugs, IMiDs; vascular endothelial growth factor, VEGF; antibody, Ab; chemokine receptor 4, CCR4; chemokine ligand 12, CXCL12; Dickkopf, DKK; receptor activator of nuclear factor-kappa-Β ligand, RANKL; transforming growth factor-beta 1, TGF-β1; tumor necrosis factor-alpha, TNF-α; interleukin-6, IL-6; programmed cell death 1/programmed cell death ligand 1, PD-1/PD-L1; chimeric antigen receptor-Tcells, CAR-Tcells; marrow inﬁltrating lymphocytes, MILs; self-ligand receptor of the signalling lymphocytic activation molecule, SLAMF7. cytokine acts as both a growth factor and a survival factor. In 5. Conclusions long-term and high cell density cultures, malignant plasma MM is likely one of the hematologic conditions in which the cells become progressively independent and are able to major advances from biology to new therapy have occurred produce IL-6 as an autocrine growth factor [155]. (ese over the last years. (e biology outlook has shifted from pieces of evidence prompted several investigations aimed at morphology and basic biochemical analysis to an integrated characterizing additional soluble substances supporting MM multi-Omics approach oﬀering novel therapeutic perspec- in the environment. TGF-β is one of the best candidates tives. Nonetheless, MM ﬁnally progresses to a relapse/re- deserving druggable intervention investigation [156], fractory stage, levying a heavy impact on patient survival and employing several blocking approaches [157, 158], dem- quality of life. (e MM microenvironment pathophysiologic onstrating clinical activity in the treatment of MM-associ- determinants, deﬁned from a validated prognostic per- ated bone diseases. Luspatercept treatment has been shown spective, provide clinicians with novel insight, oﬀering the to have a potential impact on MM-related kidney involve- potential to deal with the unmet medical need for prolonged, ment (Table 1 and Figure 2). sustained disease remission. (e interactions of multiple Ultimately, adequateoxygenpressureisessentialfor proper myeloma cells with diﬀerent subsets of immune cells and physiologic conditions and insuﬃcient hypoxia is a conspic- ECs within the BM tumor niche environment seem to be the uous characteristic in various physiological and pathological ideal backbone supporting the ultimate translation of bio- processes, including neoplastic disorders and cancer dissemi- logical ﬁndings into improved diagnostics and therapies. nation [159]. In MM, increased BM hypoxia is associated with an increased recirculation of neoplastic cells [160], leading to loss of pharmacological sensitivity and priming resistance to Conflicts of Interest radiotherapy [161]. (erefore, alkylators selectively activated by (e authors declare that they have no conﬂicts of interest. insuﬃcient oxygen supply condition were tested in preclinical models [162] and in clinical trials involving patients with MM [162, 163], showing an eﬀective inhibition of HIF-1-alpha in Acknowledgments MM both alone and in combination [164]. Intriguing results from phase I/II clinical studies results supported further in- (e authors disclose the support received by the Italian vestigation in relapsed/refractory subjects [163]. (e thera- Association for Cancer Research (AIRC) through an In- peutic opportunity window and pathophysiological aspects are vestigator Grant (no. 20441), by the GLOBALDOC Project summarized in Figure 2. to AGS. 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Publisher: Hindawi Publishing Corporation
Copyright: Copyright © 2020 Antonio Giovanni Solimando et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ISSN: 1687-8450
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DOI: 10.1155/2020/6820241
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Abstract

Hindawi Journal of Oncology Volume 2020, Article ID 6820241, 16 pages https://doi.org/10.1155/2020/6820241 Review Article A Comprehensive Biological and Clinical Perspective Can Drive a Patient-Tailored Approach to Multiple Myeloma: Bridging the Gaps between the Plasma Cell and the Neoplastic Niche 1 1 2 Antonio Giovanni Solimando, Angelo Vacca, and Domenico Ribatti Department of Biomedical Sciences and Human Oncology, Section of Internal Medicine “Guido Baccelli” University of Bari Aldo Moro, Piazza Giulio Cesare, 11, 70124 Bari, Italy Department of Basic Medical Sciences, Neurosciences and Sensory Organs, University of Bari Medical School, Bari, Italy Correspondence should be addressed to Domenico Ribatti; domenico.ribatti@uniba.it Received 21 December 2019; Revised 17 February 2020; Accepted 10 April 2020; Published 18 May 2020 Guest Editor: Klaus Podar Copyright © 2020 Antonio Giovanni Solimando et al. (is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (ere is a broad spectrum of diseases labeled as multiple myeloma (MM). (is is due not only to the composite prognostic risk factors leading to diﬀerent clinical outcomes and responses to treatments but also to the composite tumor microenvironment that is involved in a vicious cycle with the MM plasma cells. New therapeutic strategies have improved MM patients’ chances of survival. Nevertheless, certain patients’ subgroups have a particularly unfavorable prognosis. Biological stratiﬁcation can be subdivided into patient, disease, or therapy-related factors. Alternatively, the biological signature of aggressive disease and dismal therapeutic response can promote a dynamic, comprehensive strategic approach, better tailoring the clinical management of high- risk proﬁles and refractoriness to therapy and taking into account the role played by the MM milieu. By means of an extensive literature search, we have reviewed the state-of-the-art pathophysiological insights obtained from translational investigations of the MM-bone marrow microenvironment. A good knowledge of the MM niche pathophysiological dissection is crucial to tailor personalized approaches in a bench-bedside fashion. (e discussion in this review pinpoints two main aspects that appear fundamental in order to gain novel and deﬁnitive results from the biology of MM. A systematic knowledge of the plasma cell disorder, along with greater eﬀorts to face the unmet needs present in MM evolution, promises to open a new therapeutic window looking out onto the plethora of scientiﬁc evidence about the myeloma and the bystander cells. urine light chains. (ese parameters may be useful to foresee 1. Introduction the patient characteristics from a biological standpoint, in Multiple myeloma (MM) is an incurable haematological order to predict therapy response and estimate the MM load malignancy characterized by a clonal proliferation of plasma [3]. Nonetheless, the D&S is aﬀected by observer-related bias cells that accumulate preferentially in the bone marrow in quantifying lytic lesions, and since 2005, it has been (BM). It accounts for 1% of all cancers and 10% of all replaced by the International Staging System (ISS), which is haematological malignancies. Resistance to chemotherapy based only on the combination of two parameters, namely, poses one of the main challenges in MM management [1]. β2-microglobulin and albumin [4]. Nowadays, sensitivity Indeed, although advances in MM pathophysiological and speciﬁcity of bone disease identiﬁcation have improved, deconvolution and therapeutic knowledge, MM is still an thanks to the widespread use of computed tomography and incurable disease [2]. According to Durie–Salmon (D&S) of functional imaging such as magnetic resonance and clinical staging, MM patients can be stratiﬁed based on positron-emission tomography (PET scan) [1]. Moreover, available clinical parameters, such as haemoglobin, serum among the three stages, data on ISS stage III, associated with calcium value, X-ray bone study, immunoglobulins, and the poorest outcome, are now available. Since cytogenetics, 2 Journal of Oncology evaluated by ﬂuorescent in situ hybridization (FISH), is Current studies are focused on the bone marrow mi- also a major prognostic factor, a new paradigm shift in croenvironment and inﬂammatory cells as attractive drug- gable targets [15]. (e MM physiology oﬀers a wide range of patient risk stratiﬁcation has incorporated the three re- current genetic abnormalities, the t(4; 14), deletion(17p), targeting opportunities, which can be useful in chemo- and t(14; 16), that are mostly associated with a poorer therapeutics for devising more personalized therapy for MM outcome. (ese are used, along with the clinical and patients [16]. For example, increased BM hypoxia is asso- laboratory parameters, in order to gain a more reliable ciated with increased recirculation of MM plasma cells MM risk classiﬁcation according to the revised ISS (R-ISS) (MM-PCs). Oxygen delivery decrease, by enhancing hyp- [5]. Undoubtedly, in MM, the genomic landscape has a oxia-inducible factor-2 alpha (HIF-2α) activity, induces strong impact on patient outcome and response to therapy MM-PC chemokine ligand 12 (CXCL12) upregulation, with [6–8]. Nevertheless, the disease aggressiveness is not only a diminished migration toward CXCL12 and reduced ad- linked to multistep genetic events but also to the MM hesion to mesenchymal stromal cells in vitro. HIF-2α also microenvironment and the MM bystander cells, involved strongly induced the expression of chemokine receptor 1 (CCR1) in MM-PCs. CCR1 enhances MM-PC dissemina- within the tumor niche in a vicious cycle that leads to MM evolution into more complex pathological architecture tion toward CCL3, while decreasing the MM-PC motility [9]. BM microenvironment-mediated drug resistance is reaction to CXCL12. Additionally, CCR1 upregulation by the main mechanism allowing MM to evade the eﬀects of MM-PCs was correlated with a poor outcome in newly conventional and new drugs [10]. To date, a plethora of diagnosed MM subjects and associated with enhanced cir- pathophysiological mechanisms has been dissected, but culating MM-PCs in these individuals. Taken together, these potential targets considered suitable for therapeutic in- data suggest a role for hypoxia-mediated CCR1 upregulation terventions aimed at interfering with the mutual inter- in driving the egress of MM-PCs from the BM. Targeting actions between the clonal plasma cells and the tumor CCR1 may be a novel strategy to prevent dissemination and milieu have proven largely unsatisfactory [11]. In this overt relapse in MM [17]. scenario, we have carried out an extensive literature re- Mesenchymal stem cells (MSCs), one of the main cell components within the BM milieu, can disseminate toward view to probe the novel insights available from transla- tional investigations, in order to reach a deeper primary tumors and metastatic sites, implying that these understanding of the emerging therapeutic window from cells might modulate tumor growth and metastasis [13]. a bench-to-bedside standpoint. Myeloma-derived MSCs can deeply impact the disease homeostasis. (erefore, MSCs do not represent bystanders in the BM niche but rather dynamic actors in the MM 2. The Role of the Bone Marrow biology. MSCs can represent a novel target to develop the Microenvironment: Novel Molecular next generation of therapy in cancer, both by in vitro en- gineering as antitumor carrier to the tumor sites. MM is no Dependencies in Multiple Myeloma exception to this principle [18]. MSCs were lentivirally Signals from the bone marrow microenvironment play a engineered with osteoprotegerin (OPG) in preclinical pivotal role in supporting MM cell growth, spread, and models aimed to halt MM-related skeletal lesions [19]. (e survival, as well as MM progression [12]. In the bone ﬁrst-in-class proteasome inhibitor bortezomib shapes the marrow microenvironment, the cellular compartment tumor-friendly MM environment by inducing bone matrix consists of hematologic and nonhematopoietic cells such as remodelling [20] and by interfering with MSC diﬀerentia- stromal cells, ﬁbroblasts, osteoblasts, osteoclasts, endothelial tion toward the osteoblastic phenotype [21]. (erefore, cells (ECs), B cells, T cells, natural killer (NK) cells, mac- combination strategies combined proteasome inhibition rophages, mast cells, and myeloid-derived suppressor cells with both vitamin D [22] and epigenetic regulators [23]. (MDSCs). During MM development, MM cells can aﬀect the Building on these strategies, diﬀerent groups unravelled BM cells through cell-cell contact or the secretion of soluble novel mechanisms able to mobilize and eradicate niche- factors to build up a favorable microenvironment. MM cells protected myeloma cells by employing histone deacetylase adhere to bone marrow stromal cells (BMSCs) and trigger inhibitors (HDACis) [24]. Pharmacological interfering with many pathways in the latter, resulting in the transcription nucleosome conformation changes and skeletal metabolism and secretion of multiple cytokines such as interleukin-6 demonstrated the interruption of the molecular crosstalk (IL-6), insulin-like growth factor-1 (ILGF-1), vascular en- between MM cells and the stroma and uncovered indirect dothelial growth factor (VEGF), and stromal cell-derived eﬀects halting cell proliferation, bone disease, and angio- factor-1α (SDF-1α) which mediate MM cell growth, pro- genesis, in vitro and in vivo [24–26]. liferation, survival, and drug resistance [13]. Next, MM cells (e myeloma microenvironment is also characterized by educate the bone marrow cells to support neoplastic cell Notch signalling hyperactivation due to the increased ex- growth, survival, and the acquisition of drug resistance pression of Notch 1 and 2 and the ligands Jagged 1 and 2 in resulting in disease relapse, conferring a survival advantage. tumor cells. Notch activation inﬂuences myeloma cell bi- (e tumor microenvironment is recognized as one of the ology and promotes the reprogramming of bone marrow leading factors promoting chemoresistance, but the mech- stromal cells. Colombo et al. [27] uncovered Jagged blocking anisms responsible for this eﬀect are still largely obscure to be relevant for dismal sensitivity to alkylating agents, [14]. immunomodulatory drugs (IMiDs), and proteasomal Journal of Oncology 3 block MM homing and enhance drug eﬃcacy as well. In inhibition due to MM cell and tumor milieu-related mechanisms. Enhanced CXCR4/SDF-1 alpha signalling is frame of this thinking, Natoni et al. [30] have shown that BMSCs can nurse MM by shaping an immune-tolerogenic boosted by Notch overactivation within the MM environ- ment. Additionally, this chemokine axis fuels antiapoptotic milieu and uncovered sialylation as an actionable mecha- mechanisms [27], prompting therapeutic approaches nism to boost the immune response [30]. holding the potential to interrupt the vicious cycle between the tumoral PCs and the BMSCs and, conceivably, improve 2.1. Angiogenesis in Multiple Myeloma. In 1994, Vacca et al. patients’ responses to standard-of-care therapies [27]. Furthermore, CXCR4/SDF-1 alpha signalling has been [37] demonstrated for the ﬁrst time that bone marrow microvascular density (MVD) was signiﬁcantly increased in revealed to impact clinical outcome in PC dyscrasias. MM compared to monoclonal gammopathies of undeter- Nevertheless, treatment strategies pinpointing this receptor mined signiﬁcance (MGUS) and even more in active vs. or its cognate ligand (burixafor or plerixafor) deemed not nonactive forms. (e close association between angiogenesis adequately proﬁcient. (erefore, a deeper characterization and active MM indicates that it is the vascular phase of of the biological CXCL12/CXCR4 interaction can oﬀer plasma cell tumors. Conversely, MGUS and nonactive MM additional insights, overcoming PC disorder treatment re- sistance and clonal resilience. (is could allow envisioning a represent the avascular phase. (e microvessel area and the labeling index (LI) percent are closely associated with the novel therapeutic window and a more eﬀective drug com- bination, designed to halt myeloma progression [28, 29]. MM activity phase and are mutually correlated [38–41]. In 2011, Ria and colleagues [15] also highlighted neo- Additionally, glycosylation, by modulating diﬀerent aspects of tumor biology, can be considered as a hallmark of vascularization as a constant hallmark of MM progression. (is process is only partially supported by factors such as cancer. Several solid and haematological malignancies are VEGF, ﬁbroblast growth factor-2 (FGF-2), and metal- characterized by enhanced sialylated glycan expression, with loproteinases (MMPs), which are directly secreted by the a direct correlation with higher disseminated behaviour. tumor cells. As a consequence of plasma cell-stromal cell Sialylation can also prime MM homing into its environment interactions, the cytokines within the MM niche, in par- by physical interaction between skeletal precursors, stromal ticular IL-6, drive the release of angiogenic factors from cells, and MM cells creating niches and educating bone cells. (erefore, interfering with sialylation may promote trans- bystanders in the bone milieu, these being one of the main triggers of the angiogenic switch during disease progression. lational navigation of the milieu-drug resistance boundaries and deﬁne alternative combinatorial treatment strategies But along with angiogenesis, vasculogenesis also occurs in the tumor niche of MM subjects, enhancing the vascular tree bringing sialylation inhibitors to the MM-stroma interface formation. In the neoplastic microenvironment of MM [30]. (us, nanotechnologically engineered tools provided individuals, hematopoietic stem cells are primed to become next-generation strategies for tailored anti-MM therapy by ECs by the angiogenic cytokines shed in autocrine, para- optimization of pharmacokinetics and pharmacodynamics crine, and endocrine fashion. proﬁle of conventional chemotherapeutic agents [31]. In (erapeutic strategies in MM consist of conventional detail, novel anthracycline preparation integrating integrin α4β1 within nanoparticles seems to be able to exert enhanced chemotherapy and biologically based therapy targeting not only MM-PCs but also the microenvironment and angio- anti-MM and dismal oﬀ-target eﬀects, oﬀering a proof of concept of the value of this pharmacokinetics innovation genesis. Bortezomib regulates many cellular processes, in- cluding the modulation of transcription factors, such as NF- [31]. An alternative approach was delivering liposomal formulation carrying combinations of taxanes, alendronate, κB, cell cycle progression, inﬂammation, immune surveil- lance, growth arrest, and apoptosis. NF-κB is a major and isoform-adapted transferrin, enabling microenviron- transcriptional factor which mediates the expression of ment drug modulation [32]. Notably, novel engineered many proteins including cytokines, chemokines, and cell tagging strategies combined modern immunotherapeutic adhesion molecules. Bortezomib inhibits NF-κB, enhancing targeting with either proteasome inhibitor [33] or bone- the susceptibility of MM plasma cells to therapeutics, while modifying agents (BMAs) [34], gaining more eﬃcient oﬀ- the induction of IL-6 by BMSCs mediated by NF-κB in- target proﬁle. Speciﬁcally, CD38 receptor- and B-cell mat- uration antigen- (BCMA-) directed approaches have in- creases the secretion of VEGF from MM-PCs. Furthermore, bortezomib inhibits MMEC mitotic activity, through inhi- troduced a practice change in immunotherapy and are being intensively investigated in MM [34, 35], since these mole- bition of VEGF, IL-6, insulin-like growth factor-1 (IGF-1), and angiopoietin-1 and angiopoietin-2 (Ang-1 and Ang-2) cules are highly expressed on the malignant plasma cells. Sialylation inhibition using these approaches also [42]. (e circulating levels of Ang-1, Ang-2, VEGF, and promises incremental activity in interfering within the MM- angiogenin were measured in 54 patients with smouldering niche vicious cycle. Recently, sialyltransferase inhibitors MM (SMM). (is result was compared with those of 27 restored aﬀective anti-MM activity by restoring innate and MGUS patients, 55 MM patients, and 22 healthy controls, acquired immune response, while halting malignant cell demonstrating that the ratio of circulating Ang-1/Ang-2 was proliferation at the same time [36]. reduced in MM individuals with full-blown overt MM due to Currently, delivery systems employing the sialylation inhibitor 3Fax-Neu5Ac encapsulation in combination with a biologically signiﬁcant enhancement of Ang-2, but not in SMM or MGUS nonmalignant control subjects. VEGF and BMA are intensively investigated, in order to potentially 4 Journal of Oncology angiogenin were increased in all patients compared to Bisphosphonates exert a direct eﬀect on MM plasma controls. However, circulating VEGF was higher in symp- cells [55]. In detail, both zoledronic acid and neridronate tomatic MM compared to SMM and MGUS, while angio- have a cytotoxic activity on tumor cells and inhibit an- genin was reduced. Hence, the above data show that the giogenesis [55–57]. (e side eﬀect is osteonecrosis of the Ang-1/Ang-2/Tie-2 axis may be an eﬀective target for the jaw (ONJ), a long-lasting disorder that occurs mainly in development of novel antimyeloma agents [43]. Bortezomib breast cancer and MM patients treated with intravenous downregulates not only the caveolin-1 tyrosine phosphor- bisphosphonates [58]. ylation, responsible for VEGF-mediated MM cell migration, Recently, the role of CX3CL1/fractalkine has been but also the caveolin-1 phosphorylation induced by VEGF in reported, as a novel mechanism of this cell signalling ECs. Finally, bortezomib inhibits the transcription of ICAM- boosting angiogenesis and inﬂammation in multiple 1, VCAM1, and E-selectin [44]. myeloma. ADAM10 and ADAM17 are responsible for (alidomide is an antiangiogenic drug. It modulates tu- cleavage and shedding, thereby modulating CX3CL1/ mor necrosis factor-alpha (TNF-α) signalling through direct fractalkine release. Notably, these MMPs are regulated by and indirect eﬀects on the tumor microenvironment [45]. It inﬂammatory and angiogenesis processes. Firstly, as- also reduces FGF-2 [46], VEGF, and IL-6 secretion by BMSCs sessment of soluble levels in plasma cell disorders at and MM cells [47], stimulating the activation and expansion of diﬀerent disease stages demonstrated that circulating T cells and enhancing NK-cell-mediated cytotoxicity. (a- concentration of CX3CL1 was signiﬁcantly higher in full- lidomide disrupts the host marrow-MM cell interactions by blown disease compared with controls [59]. Strikingly, selectively modulating the density of cell surface adhesion this observation was correlated with BM microvessel molecules. Nonetheless, treatment with thalidomide induces density. Next, ensuing functional in vitro experiments side eﬀects while lenalidomide and pomalidomide, its deriv- recapitulated fractalkine dynamics, highlighting the atives, are both less toxic and more potent [48, 49]. Cereblon theragnostic role of enhanced production of this che- (CRBN) is a primary target of thalidomide teratogenicity, but mokine by MM-derived BM endothelium upon exoge- it is also required for the antimyeloma activity of thalidomide nous stimulus [59, 60]. In fact, Tanaka et al. [61] uncovered mAb-blocking strategies anti-CX3CL1 as next- and related drugs (IMiDs). A decreased CRBN expression is linked to pharmacological resistance in human MM cell line generation approach aimed at halting innate and adaptive models and primary cells and may also provide a biomarker to immune-dependent inﬂammation. Finally, Chen et al. predict IMiD response and resistance [49]. In fact, other also corroborated this evidence demonstrated decreased authors analysed the inﬂuence of the single-nucleotide poly- CX3CL1 production in vivo, upon proteasome inhibition morphisms (SNPs) of the CRBN gene on the risk of adverse [62]. (ese compelling data envision also the use of anti- eﬀects of thalidomide-based chemotherapy in patients with TNF-α, in combination with the abovementioned thera- MM [50]. peutic strategies for MM patients [59]. However, Curry and colleagues found no reduction in MVD before or after treatment with thalidomide of newly diagnosed MM (NDMM) patients [51]. Nevertheless, other 2.2. Endothelial Cells. MMECs express VEGF/VEGFR-2, authors showed that high MVD at diagnosis was considered FGF-2/FGF-2 receptor-2 (FGF-2R-2), and Ang-2/Tie-2 and exert an increased in vitro and in vivo angiogenic activity an independent poor prognosis factor [52]. Lamanuzzi and colleagues [53] evaluated mTOR acti- [63]. Moreover, MMECs express CXC chemokines CXCL8/ IL-8, CXCL11/interferon-inducible T-cell alpha chemo- vation in ECs from 20 patients with MGUS and 47 patients with MM and its involvement in angiogenesis. mTOR and attractant (I-TAC), CXCL12/SDF-1α, and CCL2/monocyte chemotactic protein-1 (MCP-1), which mediate plasma cell the rapamycin-insensitive companion of mammalian target of rapamycin (RICTOR), two components of mTORC2 proliferation and homing [64]. Long-term MMECs were compared with MGUSECs complex [54], were deemed signiﬁcantly elevated in MMECs compared to MGUS-ECs. (e authors uncovered mTORC2 and HUVECs as their normal quiescent counterpart [63]. MMECs but not healthy cells overexpressed endothelial expressed by MMECs to be relevant for angiogenic boosting activation markers [65]. Mechanistically, MMECs in- and found that mTOR/RICTOR targeting by siRNA and dual mTOR inhibitor PP242 reduced the MMEC angiogenic creasingly produce FGF-2, VEGF, MMP-2, and MMP-9 compared to HUVECs, conferring a growth advantage functions, including cell migration, chemotaxis, adhesion, invasion, in vitro angiogenesis on Matrigel over controls by a faster establishment of a proangio- , and cyto- genic phenotypic behavior, in terms of capillary skeleton reorganization. Additionally, in the chick embryo chorioallantoic membrane (CAM) and in Matrigel plug sprouting and net formation [65]. MMECs also boost a strong proangiogenic response in the CAM [66]. Gene assays, PP242-directed approaches demonstrated angiogenic blockade in vivo by interfering with angiogenesis. PP242 expression assays corroborated these pieces of evidence, uncovering MMECs’ phenotype to be characterized by exerted a synergistic eﬀect with IMiDs and proteasome inhibitor, suggesting that mTOR blockade can enhance the enhanced proangiogenic gene transcription, namely, VEGF, FGF isoforms, HGF, Tie2, transforming growth antiangiogenic eﬀect of these drugs. Because mTORC2 in- factor-beta (TGF-β), GRO-α chemokine, ﬁbronectin-1, volved in MM angiogenesis, dual mTOR inhibitor PP242 HIF-1α, ETS-1, ID3, and osteopontin compared to could support antiangiogenic management of MM patients HUVECs. [53]. Journal of Oncology 5 MMECs alone displayed a VEGF-dependent autocrine 2.3. Macrophages and Mast Cells. Tumor-associated mac- growth loop [65], owing to high VEGF and VEGFR-2 ex- rophages and mast cells support tumor growth and neo- vascularization by producing a wide array of angiogenic pression, constitutive autophosphorylation in both VEGFR- 2 and the associated kinase ERK-2, along with the inhibition cytokines. Mast cell- and macrophage-derived growth fac- of proliferation, capillarogenesis, and phosphorylation by tors that can promote tumor development and angiogenesis neutralizing anti-VEGF and anti-VEGFR-2 antibodies. include TNF-α, TGF-β1, FGF-2, VEGF, platelet-derived Pentraxin 3 aﬀected MMEC functional activities and was growth factor (PDGF), IL-8, osteopontin, and nerve growth able to modify the angiogenic capability of both MMECs and factor (NGF). Conversely, mast cell- and macrophage- plasma cells [67]. produced cytokines that may participate in antitumor re- Comparative gene expression proﬁling was made of sponses include IL-1, IL-2, IL-4, IL-10, and interferon- MMECs and MGUSECs with Aﬀymetrix U133A arrays gamma (IFN-c) [73]. [68]. Expression of 22 genes deemed signiﬁcantly diﬀerent When BM macrophages from MM patients are exposed by comparing MMECs with MGUSECs. Key biological to VEGF and FGF-2, they transform into cells that are functionally and phenotypically similar to paired MMECs, processes related to protumorigenic functions were af- fected, showing signiﬁcant gene expression deregulation and generate capillary-like networks mimicking those of in the symptomatic disease when compared to the pre- MMECs [74]. Macrophages from nonactive MM, MGUS, cursor’s states. Next, DIRAS3, SERPINF1, SRPX, BNIP3, and benign anaemia patients display similar albeit weaker IER3, and SEPW1, gene-encoding proteins, were func- features. EC-like macrophages and apparently typical tionally tested to substantiate the gene signature ﬁndings, macrophages contribute considerably to the formation of corroborating their proangiogenic function in BMECs. new vessels in patients with active MM, whereas their BNIP3, IER3, and SEPW1 transient gene silencing had a vascular supply is minimal in nonactive MM and absent in signiﬁcant impact on programmed death, cell prolifera- MGUS patients and control patients [74]. In contrast to tion, adhesion, and angiogenesis-related functions. Four MGUS and asymptomatic disease, CD14/CD68 surface proteins were found to be overexpressed in MMECs: overexpression has been found in full-blown myeloma. BM trephine immune staining additionally dissected two mac- ﬁlamin A, vimentin, α-crystallin B, and 14-3-3ζ/δ protein [69]. (eir expression was enhanced by VEGF, FGF-2, rophage subpopulation, demonstrating cells with either HGF, and MM-PC-conditioned medium and their si- endothelial or conventional phenotype by CD68/FVIII-RA lencing RNA knockdown aﬀected MMEC angiogenesis- coloration. Remarkably, proangiogenic eﬀects on macro- related functions, including spreading, migration, and phages have shown to contribute to the building of neovessel tubular morphogenesis [69]. wall in patients with active MM over nonactive and MGUS More recently, Leone described a novel aspect of conditions [74]. disease pathophysiology, by characterizing the MM cell Proinﬂammatory macrophages in BM biopsies are a interface with the local environment, namely, vascular potential prognostic biomarker for acquired MM resistance endothelium between ECs and CD8 lymphocyte, create a to bortezomib therapy, and high levels in BM are correlated permissive immune microenvironment within the BM, with poor survival. Remarkably, proteasome inhibitor treatment of proinﬂammatory macrophages primed MM- allowing plasma cell proliferation. In this context, the corrupted endothelium behaves as tolerogenic promoter, tumor-initiating cell (MM-TIC) inﬁltration both in vitro and in vivo in an IL-1β-dependent fashion. One way to abolish by indirect negative regulation of the eﬀector memory + + CD8 T cells. (e CD8 T-cell population sustained by bortezomib-induced MM-TIC enrichment is by blocking ECs also expressed Foxp3, producing IL-10 and TGF-β, the IL1β axis using a pharmacologic or genetic approach and exerting a protumorigenic activity. (e above study [75]. Additionally, CD163 expression was detected by im- adds further insight into the role that ECs play in MM munohistochemistry to determine the number of tumor- biology and describes an additional immune regulatory associated macrophages (TAMs) in 198 MM patients re- mechanism that inhibits the development of antitumor ceiving bortezomib-based regimens. Enhanced CD163+ TAM inﬁltration in NDMM was correlated with a worse immunity and may impair the success of cancer immu- notherapy [70]. clinical outcome, in terms of progression-free survival (PFS), overall survival (OS), and a worse therapy response quality Extracellular vesicles (EVs) shed from the MM cell surface actively participate in cellular crosstalk and vessel compared with subjects with lower CD163+ TAM inﬁltra- tion. (ese data indicate that the CD163+ TAM content at formation during MM progression [71]. Proteasome inhi- bition via Bi-EVs decreased EC proteasome activity, and Bi- diagnosis is a powerful predictor of prognosis in MM [76]. EVs released from apoptotic MMECs promoted angiogen- Another link between the eﬀect of bortezomib in MM pa- esis suppression by decreasing the proliferation and mi- tients and macrophages was highlighted by Khalife’s studies. gration of ECs [71] IMiDs exerted a relevant antiangiogenic (ey demonstrated the improved treatment eﬀectiveness eﬀect in vivo, and in vitro, it also inhibited migration of gained by miR-16 increased expression in boosting anti-MM MMECs, but not of MGECs or control HUVECs. VEGF/ activity by a proteasome inhibitor in the presence of MM resident TAMs [77]. Enhanced soluble miR-16 in MM in- VEGFR-2 cell signalling was deemed biologically connected to lenalidomide treatment, which exerted a signiﬁcant im- dividuals linked to more favourable outcome. Conversely, deletion 13 on cancer cells was inversely associated with pact on cytoskeleton rearrangement, migration, and cell metabolism in MMECs [72]. peripheral miR-16 concentration [78]. miR-16 can be 6 Journal of Oncology Additionally, urinary N-terminal propeptide of procollagen actively secreted by MM cells through EVs, with a direct correlation between intracellular and shed levels. EVs iso- type I (Ntx) concentrations was assessed by an enzyme- linked assay, at diﬀerent disease stages and bone involve- lated from MM patients can drive circulating monocyte diﬀerentiation to M2-TAMs, while the increased concen- ment. Enhanced mast cell count, RANKL, and Ntx con- tration of circulating miR-16 reverts this behaviour. In vivo, centrations were found in MM subjects. Furthermore, mast miR-16 lost sustains macrophage diﬀerentiation toward an cell density was positively correlated with MMP-9, RANKL, M2 phenotype acquisition, most likely due to dismal NF-κB and Ntx. (erefore, mast cells may contribute to osteolytic activation via IKKα/β targeting [77]. Moreover, the immune processes during MM progression [88]. function of macrophages is mediated by IL-32c, which is overexpressed in MM patients and associated with a more 2.4. Cancer-Associated Fibroblasts (CAFs) in MM. In the advanced clinical stage [79]. Gene expression proﬁling progression of the disease from MGUS to MM, a ﬁbroblast showed a signiﬁcant IL-32c-dependent induction of the switch is required to acquire protumorigenic activity and immunosuppressive molecule indoleamine 2,3-dioxygenase parallels the behaviour exhibited by other haematological (IDO) in macrophages, and this eﬀect was veriﬁed by qRT- and solid cancers [89–92]. (e switch was demonstrated by PCR, western blotting, and immunoﬂuorescence. Proteinase the bone marrow ﬁbroblast gene expression proﬁle of pa- 3 (PR3), an IL-32 binding protein, was universally expressed tients with MGUS and MM, extracted by nonnegative matrix on the surfaces of macrophages, and PR3 knockdown or the factorization (NMF) [93]. Moreover, a speciﬁc miR proﬁle in inhibition of the STAT3 and NF-κB pathways hindered the BM ﬁbroblasts is linked to the transition from the asymp- IL-32-gamma-mediated stimulation of IDO expression. tomatic to the full-blown disease. BM ﬁbroblasts and EV- (ese results indicate that MM cell-derived IL-32c promotes dependent vicious cycle orchestrated by MM cells determine the immunosuppressive function of macrophages and is a an enhanced production of miR-27b and miR-214, fuelling potential target for MM treatment [80]. proliferative and antiapoptotic pathways. (ese prosurvival MGUS and smouldering disease seem to be character- functions parallel an increased expression of ﬁbroblast ac- ized by a peak of mast cell density count-related and an- tivation markers alpha-smooth muscle actin (αSMA) and giogenesis enhancement [81]. Ang-1 is a crucial promoter of ﬁbroblast activation protein (FAP). While strengthening the MM cell growth by stimulating angiogenesis. Experimental mechanisms involved in the transition from MGUS and evidence indicates that Ang-1 secreted by primary murine SMM to MM, a peculiar miRNA proﬁle in MM-associated mast cells promotes marked neovascularization in an in vivo ﬁbroblasts, along with the myeloma cells, educates the BM transplantation assay [82]. Primary mast cells accelerate microenvironment by priming the BM ﬁbroblast phenotype tumor growth by established plasmacytoma cell lines, while [94]. In fact, Desantis et al. studied the eﬀect of recombinant Ang-1-neutralizing antibodies signiﬁcantly reduced the human erythropoietin (rHuEPO) on MM ﬁbroblasts in vivo growth of plasmacytomas containing mast cells. Moreover, and in vitro. It had previously been demonstrated that mast cell inﬁltrate parallels proangiogenic cytokine con- rHuEPO regulated angiogenic responses in MM via a direct centration, growth-related oncogene-alpha (GRO-alpha), eﬀect on macrophages and ECs. Likewise, rHuEPO de- and epithelial neutrophil-activating protein-78 (ENA-78). creases the activation marker (αSMA and FAP) expression (e authors also demonstrated that mast cell density was in MGUS and MM; furthermore, proinﬂammatory and correlated with ki-67 PI, suggesting an important partici- proangiogenic cytokines, such as IL-6 and IL-8, VEGF-A, pation of mast cells in MM biology and growth [83]; in this FGF-2, and HGF in MM ﬁbroblasts, signiﬁcantly dimin- context, mast cells would enhance angiogenesis, produce ished. Collectively, rHuEPO halted the MM-associated ﬁ- cytokines with growth eﬀects on myeloma cells, and modify broblast proliferation. Conversely, ﬁbroblast-programmed the BM microenvironment [84]. (erefore, mast cells could cell death enhanced in both MGUS and MM. Overall, these be indicators of the disease activity [85] and valuable targets data pinpoint rHuEPO as a key brake on MM-supporting for therapeutic interventions [83]. In line with this view- ﬁbroblast action [95]. point, mast cells may be a novel target for an adjuvant strategy aimed at halting angiogenesis by interrupting the vicious cycle underlying cytotoxic cytokine production, thus 2.5. Myeloid-Derived Suppressor Cells. MDSCs are myeloid circumventing mast cell-mediated immune suppression cells with a speciﬁc inhibitory activity on the immune re- [86]. sponse, which accumulate in the tumor microenvironment BM specimens from active myeloma over premalignant during tumor development [96] Signiﬁcant accumulations derived trephines analysed by both laser and electron mi- of immunosuppressive MDSCs were observed in the BM of croscopy were characterized by the presence of neovessels patients at early stages of MM and regulated MM growth by lined by granulated mast cells [87]. Otherwise, in MGUS, inhibiting T cells [97]. Moreover, murine MM cells directly mast cells are localized on the abluminal side of neovessels activate BM MDSCs and enhance their immunosuppressive function through soluble factors such as granulocyte-mac- [87]. However, mast cell density has an impact not only on angiogenesis but also on the progression of bone disease in rophage colony-stimulating factor (GM-CSF), promoting the immune escape of MM cells [98]. An increase of bone MM patients. In 52 MM patients, BM mast cell density was measured by immunohistochemical staining for tryptase, marrow MDSCs was also detected in the 5T33 MM mouse and serum levels of MMP-9 and RANKL were measured by a model after inoculation with MM cells [98]. In the BM of solid-phase sandwich enzyme-linked immunosorbent assay. 5T33 MM mice, exosomes derived from MM cells increased Journal of Oncology 7 the number of BM MDSCs in vivo and induced changes in dendritic cells shaping within the tumor niche and opening MDSC subpopulations which are similar to their phenotype, novel therapeutic windows [110–112]. All-trans retinoic acid (ATRA) indeed enhanced MAPK activation with dismal suggesting the involvement of exosomes in the accumulation of MDSCs [99]. reactive oxygen species levels, prompting mature myeloid MDSCs can mediate the suppression of myeloma-spe- lineage fuelling [113, 114]. ciﬁc T-cell responses through the induction of T-cell anergy Despite the existence of a correlation between MDSC and Treg development in the MM microenvironment [100]. pathophysiology and proangiogenic factors, VEGF-blocking Polymorphonuclear (PMN)-MDSCs and neutrophils mAb strategies did not succeed. Furthermore, the likelihood of equivalently sustain MM resistance to alkylators and MDSC-induced reduced sensitivity to the antiangiogenetic doxorubicin, by mediating soluble factor production. Tar- therapy discouraged further attempts in this direction [115]. geting PMN-MDSCs could enhance chemotherapy eﬃcacy Conversely, promising results generated by investigating in MM. It is well accepted that targeting MDSCs in cancer miRNA-based approaches [112, 116] hold the potential to improves the immune response and increases the eﬃcacy of reduce MM disseminated potential and provided the bases to revealMDSC-relatedtargetstoidentify,mobilize,anderadicate immunotherapy. MDSCs play an ancillary role as a suitable target to overcome MM drug resistance, an important niche-protected cells likely able to favor MM progression [117]. ﬁnding in light of recent data suggesting the beneﬁt of Aiming to implement MDSC-dependent immunosup- combined chemo- and immunotherapy treatment protocols pression halting strategies, several attempts have been made [100–102]. Due to the loss of equilibrium in the MM im- to interfere with cyclooxygenase-2 (COX-2), arginase-1 mune landscape, immune checkpoint targeting agents have expression, and inducible nitric oxide synthases and to not shown clinical activity in MM. It is therefore critically decrease reactive oxygen species production and provided important to deal with immunosuppressive mechanisms and undeniable rational for the novel association of anti-in- improve immune responses, especially in advanced MM ﬂammatory compound to the MM therapeutic backbone, in patients. New immunotherapeutic strategies such as im- order to expand the eﬀectiveness of immunotherapy and to munomodulatory drug-intensiﬁed monoclonal antibody decrease the myeloid-derived population in the MM envi- ronment [118]. Remarkably, the antigen-presenting cell treatment, immune checkpoint inhibitors, and chimeric antigen receptor T-cell therapy targeting B-cell maturation (APC) capacity of dendritic cells and ECs [76] can also open antigen have been employed in advanced-stage MM patients a further therapeutic window, since several examples have [103]. An association between high PMN-MDSC levels and recently been published, highlighting the tight crosstalk poor overall survival in MM patients has been validated. between the immune microenvironment gene signature, PMN-MDSCs induced piRNA-823 upregulation, which in vascular cells, and molecular targets, in both haematological turn enhanced DNA methylation, thus stimulating the MM and solid cancers [119, 120]. (ese studies point to the WNT cell clonal evolution. Silencing of piRNA-823 in MM cells pathway as a druggable, theragnostic marker with a plethora reduced the stemness of MSCs maintained by granulocytic of eﬀects on the immune microenvironment in cancer [121]. (G)-MDSCs, resulting in a decreased tumor burden and (is complex scenario fostered an intensive translational angiogenesis in vivo [104]. It has also been demonstrated investigation aimed at improving MM immune equilibrium that the proinﬂammatory cytokine IL-18 is critically in- lost via MDSC targeting. Other noteworthy aspects are volved in MM and its levels are associated with MDSCs. IL- related to the immune microenvironmental landscape and 18-deﬁcient mice were remarkably protected from MM its modulation through ﬂuoropyrimidine, nucleoside ana- progression in a CD8 T-cell-dependent manner. Within the logues, and anthracyclines [122]. Nonetheless, state-of-the- BM milieu, IL-18 stimulates MDSCs, sustaining MM pro- art development [123, 124] holds great potential in cir- gression. High levels of BM plasma IL-18 were associated cumventing myeloid-derived immune suppression by in- with poor survival in MM patients. (e above preclinical terfering with critical signals, such as IL-4 receptor α (IL- studies suggested that IL-18 could be a potential therapeutic 4Rα), thus reducing cell proliferation. Moreover, the binding target in MM [105]. Additionally, the estrogen eﬀect in of the aptamer to its speciﬁc receptor led to MDSC depletion and tumor growth. Peptide enrichment in both M- and hematologic malignancies including MM was studied, and treatment with 17beta-estradiol signiﬁcantly promoted the G-MDSCs by phage-dependent strategies led to the devel- opment of a peptibody, via the fusion of peculiar peptide progression of the disease. However, this eﬀect has not been attributed to a direct eﬀect of estrogen on MM cells but was sequences with the Fc portion of murine IgG2b monoclonal antibodies, demonstrating in vivo activity [124]. considered to be mediated through estrogen-induced al- terations in the tumor microenvironment. In particular, it (e main complexities of the MM microenvironment signiﬁcantly increased the ability of MDSCs to suppress cell network are summarized in Figure 1. T-cell proliferation [106]. Multiple myeloma, MM; vascular endothelial growth Botta et al. [107] pointed out developments in MDSC- factor, VEGF; nerve growth factor, NGF; ﬁbroblast growth directed approaches, by suggesting applications toward factor, FGF; interleukin, IL; tumor necrosis factor-alpha, histone-deacetylase inhibitors in MM and uncovered sig- TNF-α; transforming growth factor-beta 1, TGF-β1; platelet- derived growth factor, PDGF; hypoxia-inducible factor-1 nalling pathways involving MDSCs [108, 109], able to halt inﬂammation, by impairing JAK/STAT downstream. Epi- alpha, HIF-1α; runt-related transcription factor, RUNXs; monocyte chemotactic protein 1, MCP 1; insulin-like growth genetic modulation reduced cell ability of monocytic phe- notype granulocytic shift, by promoting macrophages or factor 1, IGF-1; stromal cell-derived factor 1, SDF-1; MIP1; OPG Osteoclasts activated 8 Journal of Oncology VEGF, FGF-2, ANG1, Angiogenesis MM cells HIF-1α MMP, HGF-SF, Endothelial cells VEGF, NGF, RUNXs TGF-β, TNF-α FGF-2, IL-8 TNF-α, Macrophage TGF-β1 VEGF, FGF-2, MCP1 VEGF, FGF-2, MMP Ostepontin IGF-1, SDF1, MIP1 CXC chemokines PDGF DKK1 HGF sclerostin Mesenchymal cell Mast cell WNT BMP4, TGF-β, bFGF Proteinase MMPs VEGF, HGF, bFGF, IL-1, IL-6, TNF-α, VEGF, HGF, Dissemination PTHrP, MCFs, MIP1α IL-6, TNF-α and spreading of VEGF, TGF-β MM cells TNF-α VEGF, HGF, TGF-β, BAFF IL-6, MMPs, IL-1β, SID1α, TNF-α RANKL RANK Stromal cells, IL-6, IL-3 ,OPG, MIPα ﬁbroblasts SID1α, TNF-α, IL-1β Osteoclast IL-6 progenitor VEGF, TNF-α Immunity deﬁciency Bone remodeling MDSCs Monocyte CD8+T cell NK cell Dendritic cell Figure 1: Soluble factors and adhesion-related interactions between MM cells and bone marrow immune microenvironment: MM-PCs prime the tumoral milieu via a plethora of mechanisms. Immune cell function, by deﬁciency in adaptive and innate response dysfunction, and proinﬂammatory cytokine production drive essential signals for microenvironment colonization and interactions. Moreover, MM-PCs control neoplastic survival and dormancy, modulating the response of the BM microenvironment cells to MM dissemination. Bone disease in MM is a prototypical malignant bone microenvironment pathologic condition. By tackling the knowledge gap on skeletal dissemination and disruption and cell-cell and cell-matrix interaction, the prevention and cure of MM progression may be better understood and targeted by immunomodulation, using combinations of MM-PC-directed agents against novel therapeutic targets. angiopoietin 1, ANG1; metalloproteinases, MMP; hepato- evolution uncovered a reciprocal crosstalk between MM cyte growth factor/scatter factor (HGF/SF), chemokine, cells with the surrounding milieu, and compelling veriﬁ- CXC; Dickkopf, DKK; wingless-type MMTV integration site cation designates that angiogenesis and immunosuppres- family, WNT; bone morphogenetic protein-4, BMP4; sion often fuel a simultaneous vicious cycle [70]. parathyroid hormone-related protein, PTHrP; macrophage Consequently, approaches relating to antiangiogenic im- colony-stimulating factors, MCFs; B-cell activating factor, mune mechanisms seem to hold the promise to tip the BAFF; SID1 transmembrane family, member 1, SID1; re- equilibrium of the MM environment and increase clinical ceptor activator of nuclear factor-kappa-Β ligand, RANKL; beneﬁt. (e ﬁrst-in-class drug thalidomide and its deriv- osteoprotegerin, OPG; natural killer, NK; myeloid-derived ative lenalidomide mirrored the abovementioned knowl- suppressor cells, MDSCs. edge, representing one possible translation from bench-to- bedside eﬀorts [51, 126]. But as stated above, the precise target of lenalidomide is cereblon, since low cereblon levels 3. Therapeutic Windows are associated with drug resistance [127–129]. Undeniable A new goal in haematological malignancies is represented by evidence supports the use of drugs that target the BM a treatment approach targeting not only patients with active microenvironment to prevent the progression of SMM or MM, but also those with SMM. (is shift toward early full-blown MM. Additionally, in a mouse model, the use of intervention [125] with the antiangiogenic agents lenali- an antiangiogenic anti-VEGFR-2 antibody in the early domide and dexamethasone demonstrated prolonged dis- stage delayed tumor progression of MM; nonetheless, ease-free survival and OS in patients with SMM. besides IMiDs, angiogenic-directed strategy did not show eﬀective results in the unselected patient subgroup in patients with MM [130]. In order to achieve a patient- 4. Targeting Angiogenesis and the Immune tailored vasculogenic targeting, stringent patient stratiﬁ- Microenvironment in Multiple Myeloma: cation has been proposed by modulating from the critical Current Challenges step of MM evolution that can be critically dependent on vessel supply, such as smouldering phases [131] or extra- (e knowledge of critical pathways supporting angiogen- medullary dissemination [132–134]. esis and creating immunosuppression during MM Osteoblasts Journal of Oncology 9 Table 1: Compounds in advanced investigation targeting MM cells and the tumor microenvironment. Targets (erapeutic agents ∗ ∗ (1) IMiDs (thalidomide , lenalidomide ) Angiogenesis (2) Anti-VEGF Ab (bevacizumab) (3) Tyrosine kinase inhibitor against VEGF (sunitinib) Hypoxia (1) Evofosfamide investigational hypoxia-activated prodrug (1) CXCR4 inhibitors (plerixafor) Stromal cells (2) Anti-CXCR4 Ab (ulocuplumab) (3) CXCL12 inhibitor (olaptesed pegol) (1) Bisphosphonates (2) Anti-DKK1 Ab Osteoclasts and osteoblast (3) Anti-RANKL Ab (denosumab ) (4) Antisclerostin mAb (1) TGF-β antagonists (sotatercept) (2) TNF-α antagonists (etanercept) Cytokine signalling (3) Anti-TNF-α Ab (inﬂiximab) (4) IL-6 antagonist (siltuximab) (1) Anti-PD-1/PD-L1 Ab (pembrolizumab, nivolumab) (2) Anti-CTLA4 Ab (ipilimumab) (3) CAR-T cells Stimulation of anticancer immunity (4) MILs (5) Vaccines (6) Anti-CD38 Ab (daratumumab , isatuximab) (7) Anti-SLAMF7 Ab (elotuzumab ) Immunomodulatory drugs, IMiDs; vascular endothelial growth factor, VEGF; antibody, Ab; chemokine receptor 4, CCR4; chemokine ligand 12, CXCL12; Dickkopf, DKK; receptor activator of nuclear factor-kappa-Β ligand, RANKL; transforming growth factor-beta 1, TGF-β1; tumor necrosis factor-alpha, TNF- α; interleukin-6, IL-6; programmed cell death 1/programmed cell death ligand 1, PD-1/PD-L1; chimeric antigen receptor-T cells, CAR-T cells; marrow inﬁltrating lymphocytes, MILs; self-ligand receptor of the signalling lymphocytic activation molecule, SLAMF7. FDA and EMA approved. (e next breakthrough of therapeutic strategy design is addition, studies of chimeric antigen receptor-T-cell (CAR-T- targeting the MM ecosystem together with the immune cell) therapy targeting BCMA have revealed very high re- microenvironment. (e altered BM niche sustains the pro- sponse rates in heavily pretreated patients with MM [140, 144]. liferation of MM cells, nursed by physical and soluble re- ciprocal interactions educating both the neoplastic and the As pointed out, CXCL12 and its ligand CXCRA can have immune environmental cells [135]. Identiﬁcation, mobiliza- a pathologic role in diﬀerent stages of MM and patient drug tion, and eradication of this niche-protected dormant and resistance, so disrupting the CXCL12-CXCR4 axis might be often pharmacological insensitive cells have been signiﬁcantly a therapeutic opportunity [39]. Roccaro et al. uncovered improved since several trialsinvolving antibodies have proved CXCL12 and CXCR4 as putative targets to halt MM evo- clinical beneﬁts in MM. Anti-SLAMF7 targeting by elotu- lution and extramedullary dissemination in animal models zumab anti-CD38-directed approaches by daratumumab as a [28, 145], indicating broad potential consequences on ad- single agent or with proteasome inhibitor and IMiDs have hesion-mediated MM dissemination [29, 101, 146] and drug shown far more eﬀectiveness and superior activity than the resistance, as in other solid and haematological malignancies standard of care [136, 137]. (e ﬁrst-in-class SLAMF7 tar- [147–149] and prompt clinical validation [150]. (erapeutic interventions with burixafor or plerixafor (CXCR4 antag- geting molecule stimulates NK cells and macrophages; con- versely, CD38-targeting by daratumumab induces the onists) in MM are not eﬃcient enough [39]. immune system triggering toward Treg reduction and by Bisphosphonates (pamidronate or zoledronic acid) [56] enhancing T-helper and cytotoxic lymphocytes [138]. An- and other BMAs, such as DKK1 inhibitors (Dickkopf WNT other target studied especially in melanoma, lung cancer, and signalling pathway inhibitors), antisclerostin mAb [151], and Hodgkin lymphoma is programmed cell death 1 (PD-1) RANKL inhibitors (denosumab) [152], represent an example [139, 140]. In more detail, the pieces of evidence that PD-1/ of attempts to target the disseminated and localized bone PD-ligand 1 (PD-L1) modulation increases T- and NK cell disease eﬀect due to the spreading of malignant plasma cells. antimyeloma eﬀects prompted the use of immune checkpoint Nonetheless, despite encouraging clinical outcome gained in full-blown disease [153], to date no clinical evidence dem- inhibitors in clinical studies. Nevertheless, the anti-PD-1/PD- L1 mAb as a single agent did not provide suﬃcient results. onstrated a survival beneﬁt by treating the asymptomatic version of myeloma [154]. Lack of inﬁltrating eﬀector cells within the MM milieu can explain the modest eﬃcacy demonstrated by these clinical Studies conducted on MM cell lines have shown that the trials [141]. (erefore, drug combination strategies encour- addition of exogenous IL-6 is essential for obtaining the aged clinical trials (NCT02289222 and NCT02331368), growth of neoplastic cells in vitro. By removing IL-6 from uncovering encouraging medical response [142, 143]. In short-term cultures, MM cells die, demonstrating that this Osteoclasts 10 Journal of Oncology Targeting hypoxia Evofosfamide Targeting cytokine signalling TGF-β antagonists (sotatercept) TNF-α antagonists (etanercept) Hypoxia Anti-TNF-α Ab (inﬂiximab) Targeting angiogenesis Endothelial cells IL-6 antagonists (siltuximab) IMiDs (talidomide, lenalidomide) Anti-VEGF Ab (bevacizumab) Angiogenesis Mast cells sunitinib (tyrosine kinase inhibitor againstVEGF) CD8+T cells Targeting stromal cells activity Stimulation of anticancer immunity Anti-PD1/PDL1 Ab CXCR4 inhibitors (plerixafor) Macrophages (pembrolizumab, nivolumab) Anti-CXCR4 Ab (ulocuplumab) CXCL12 inhibitors(olaptesed Anti-CTLA4 Ab (ipilimumab) CAR-T cells pegol) Stromal cells, MILs ﬁbroblasts Vaccines Dendritic Anti-CD38 Ab (daratumumab) cells Anti-SLAMF7 Ab (elotuzumab) Targeting osteoclasts and osteoblasts NK cells Bisphosphonates Anti-DKK1 Ab Anti-RANKL Ab (denosumab) MDSCs Antisclerostin mAb Figure 2: (erapeutic targets within the tumor milieu in multiple myeloma. Immunomodulatory drugs, IMiDs; vascular endothelial growth factor, VEGF; antibody, Ab; chemokine receptor 4, CCR4; chemokine ligand 12, CXCL12; Dickkopf, DKK; receptor activator of nuclear factor-kappa-Β ligand, RANKL; transforming growth factor-beta 1, TGF-β1; tumor necrosis factor-alpha, TNF-α; interleukin-6, IL-6; programmed cell death 1/programmed cell death ligand 1, PD-1/PD-L1; chimeric antigen receptor-Tcells, CAR-Tcells; marrow inﬁltrating lymphocytes, MILs; self-ligand receptor of the signalling lymphocytic activation molecule, SLAMF7. cytokine acts as both a growth factor and a survival factor. In 5. Conclusions long-term and high cell density cultures, malignant plasma MM is likely one of the hematologic conditions in which the cells become progressively independent and are able to major advances from biology to new therapy have occurred produce IL-6 as an autocrine growth factor [155]. (ese over the last years. (e biology outlook has shifted from pieces of evidence prompted several investigations aimed at morphology and basic biochemical analysis to an integrated characterizing additional soluble substances supporting MM multi-Omics approach oﬀering novel therapeutic perspec- in the environment. TGF-β is one of the best candidates tives. Nonetheless, MM ﬁnally progresses to a relapse/re- deserving druggable intervention investigation [156], fractory stage, levying a heavy impact on patient survival and employing several blocking approaches [157, 158], dem- quality of life. (e MM microenvironment pathophysiologic onstrating clinical activity in the treatment of MM-associ- determinants, deﬁned from a validated prognostic per- ated bone diseases. Luspatercept treatment has been shown spective, provide clinicians with novel insight, oﬀering the to have a potential impact on MM-related kidney involve- potential to deal with the unmet medical need for prolonged, ment (Table 1 and Figure 2). sustained disease remission. (e interactions of multiple Ultimately, adequateoxygenpressureisessentialfor proper myeloma cells with diﬀerent subsets of immune cells and physiologic conditions and insuﬃcient hypoxia is a conspic- ECs within the BM tumor niche environment seem to be the uous characteristic in various physiological and pathological ideal backbone supporting the ultimate translation of bio- processes, including neoplastic disorders and cancer dissemi- logical ﬁndings into improved diagnostics and therapies. nation [159]. In MM, increased BM hypoxia is associated with an increased recirculation of neoplastic cells [160], leading to loss of pharmacological sensitivity and priming resistance to Conflicts of Interest radiotherapy [161]. (erefore, alkylators selectively activated by (e authors declare that they have no conﬂicts of interest. insuﬃcient oxygen supply condition were tested in preclinical models [162] and in clinical trials involving patients with MM [162, 163], showing an eﬀective inhibition of HIF-1-alpha in Acknowledgments MM both alone and in combination [164]. Intriguing results from phase I/II clinical studies results supported further in- (e authors disclose the support received by the Italian vestigation in relapsed/refractory subjects [163]. (e thera- Association for Cancer Research (AIRC) through an In- peutic opportunity window and pathophysiological aspects are vestigator Grant (no. 20441), by the GLOBALDOC Project summarized in Figure 2. to AGS. (is research project was supported in part by the Osteoblasts Journal of Oncology 11 multiple myeloma,” American Journal of Blood Research, Apulian Regional Project “Medicina di Precisione” to AGS. vol. 1, no. 1, pp. 76–89, 2011. (e authors thank SMART (Servier Medical Art, http:// [16] T. Harding, L. Baughn, S. Kumar, and B. Van Ness, “(e smart.servier.com/) for providing comprehensive medical future of myeloma precision medicine: integrating the and biological ﬁgures and fruitful datasets for the interna- compendium of known drug resistance mechanisms with tional scientiﬁc community. (e authors also thank Mary emerging tumor proﬁling technologies,” Leukemia, vol. 33, Victoria Pragnell, BA, from the University of Bari Medical no. 4, pp. 863–883, 2019. School for linguistic revision. [17] K. Vandyke, M. N. Zeissig, D. R. 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Journal

Journal of Oncology – Hindawi Publishing Corporation

Published: May 18, 2020

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Cancer associated fibroblasts and tumor growth: focus on multiple myeloma,

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Bone marrow fibroblasts overexpress miR-27b and miR-214 in step with multiple myeloma progression, dependent on tumour cell-derived exosomes,

M. A. Frassanito, V. Desantis, L. Di Marzo et al.
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Targeting B-cell non Hodgkin lymphoma: new and old tricks,

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Immunopathogenesis and immunotherapy of multiple myeloma,

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Dysregulated IL-18 is a key driver of immunosuppression and a possible therapeutic target in the multiple myeloma microenvironment,

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P. Neri, P. Tagliaferri, M. T. Di Martino et al.
New insights into the treatment of multiple myeloma with histone deacetylase inhibitors,

M. Cea, A. Cagnetta, M. Gobbi et al.
Epigenetic silencing of retinoblastoma gene regulates pathologic differentiation of myeloid cells in cancer,

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Myeloid derived suppressor cells-An overview of combat strategies to increase immunotherapy efficacy,

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Targeting the interleukin-6/Jak/stat pathway in human malignancies,

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All-trans-retinoic acid eliminates immature myeloid cells from tumor-bearing mice and improves the effect of vaccination,

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Myeloid-derived suppressor cells as regulators of the immune system,

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miR-29b induces SOCS-1 expression by promoter demethylation and negatively regulates migration of multiple myeloma and endothelial cells,

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Myeloid derived suppressor cells are numerically, functionally and phenotypically different in patients with multiple myeloma,

J. Favaloro, T. Liyadipitiya, R. Brown et al.
COX-2 inhibition improves immunotherapy and is associated with decreased numbers of myeloid-derived suppressor cells in mesothelioma. Celecoxib influences MDSC function,

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Gene expression comparison between the lymph node-positive and -negative reveals a peculiar immune microenvironment signature and a theranostic role for WNT targeting in pancreatic ductal adenocarcinoma: a pilot study,

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Wnt signalling in stem cells and cancer,

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Dendritic cells accumulate in the bone marrow of myeloma patients where they protect tumor plasma cells from CD8+ T-cell killing,

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Chemotherapeutic targeting of myeloid-derived suppressor cells,

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Generation of a new therapeutic peptide that depletes myeloid-derived suppressor cells in tumor-bearing mice,

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Lenalidomide plus dexamethasone for high-risk smoldering multiple myeloma,

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The anti-cancer drug lenalidomide inhibits angiogenesis and metastasis via multiple inhibitory effects on endothelial cell function in normoxic and hypoxic conditions,

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CIC mutation as a molecular mechanism of acquired resistance to combined BRAF-MEK inhibition in extramedullary multiple myeloma with central nervous system involvement,

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The cancer cell adhesion resistome: mechanisms, targeting and translational approaches,

E. Dickreuter and N. Cordes
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R. Vij, N. Horvath, A. Spencer et al.
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G. D’Arena, P. G. Gobbi, C. Broglia et al.
Role of interleukin-6 in the pathogenesis of multiple myeloma,

K. Gadó, G. Domján, H. Hegyesi, and A. Falus
Halting pro-survival autophagy by TGFβ inhibition in bone marrow fibroblasts overcomes bortezomib resistance in multiple myeloma patients,

M. A. Frassanito, K. De Veirman, V. Desantis et al.
Sotatercept in patients with osteolytic lesions of multiple myeloma,

K. M. Abdulkadyrov, G. N. Salogub, N. K. Khuazheva et al.
Activin receptor ligand traps in chronic kidney disease,

W. Jelkmann
Hypoxia and the extracellular matrix: drivers of tumour metastasis,

D. M. Gilkes, G. L. Semenza, and D. Wirtz
Hypoxia promotes dissemination and colonization in new bone marrow niches in waldenström macroglobulinemia,

B. Muz, P. de la Puente, F. Azab, I. M. Ghobrial, and A. K. Azab
Metabolic signature identifies novel targets for drug resistance in multiple myeloma,

P. Maiso, D. Huynh, M. Moschetta et al.
Targeting the multiple myeloma hypoxic niche with TH-302, a hypoxia-activated prodrug,

J. Hu, D. R. Handisides, E. Van Valckenborgh et al.
A phase I/II study of evofosfamide, a hypoxia-activated prodrug with or without bortezomib in subjects with relapsed/refractory multiple myeloma,

J. P. Laubach, C.-J. Liu, N. S. Raje et al.
Synergistic induction of apoptosis in multiple myeloma cells by bortezomib and hypoxia-activated prodrug TH-302, in vivo and in vitro,

J. Hu, E. Van Valckenborgh, D. Xu et al.

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A Comprehensive Biological and Clinical Perspective Can Drive a Patient-Tailored Approach to Multiple Myeloma: Bridging the Gaps between the Plasma Cell and the Neoplastic Niche

A Comprehensive Biological and Clinical Perspective Can Drive a Patient-Tailored Approach to Multiple Myeloma: Bridging the Gaps between the Plasma Cell and the Neoplastic Niche

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A Comprehensive Biological and Clinical Perspective Can Drive a Patient-Tailored Approach to Multiple Myeloma: Bridging the Gaps between the Plasma Cell and the Neoplastic Niche

A Comprehensive Biological and Clinical Perspective Can Drive a Patient-Tailored Approach to Multiple Myeloma: Bridging the Gaps between the Plasma Cell and the Neoplastic Niche

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