Supplementary Figure S1. No effects on MMECs apoptosis and proliferation by the cMET inhibition.

Supplementary Figure S2. SU11274 dose finding.

PDF file, 104K, Effect of SU11274 treatment on cMET/phospho(p)-cMET expression in R5 vs. RPMI8226 cells; and plasma cells (PCs) from relapsed/resistant vs. newly diagnosed MM patients.

Supplementary Table 2. Proteins identified in MMECs upon treatment with SU11274 or SU11274 plus bortezomib or plus lenalidomide (MALDI-TOF/TOF mass spectrometry).

Supplementary Figures S1-S4; Supplementary Table S1.

Supplementary Table 1. Real time RT-PCR primers.

Increasing evidences suggest several biological roles for erythropoietin and its receptor (Epo and EpoR), unrelated to erythropoiesis, including angiogenesis. Here, we detected the expression of EpoR in bone marrow-derived endothelial cells from monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patients (MGECs and MMECs, respectively) and assessed whether Epo plays a role in MGECs-and MMECs-mediated angiogenesis. We show that EpoR is expressed by both MGECs and MMECs even though at a higher level in the first ones. Both EC types respond to rHuEpo in terms of cell proliferation, whereas other responses, including activation of JAK2/STAT5 and PI3K/Akt pathways, cell migration and capillarogenesis are enhanced by Epo in MGECs, but not in MMECs. In addition, the conditioned media of both Epo-treated cells induce a strong angiogenic response in vivo in the chorioallantoic membrane assay, comparable to that of vascular endothelial growth factor (VEGF). Overall, these data highlight the effect of Epo on MGECs-and MMECs-mediated angiogenesis: MGECs are more responsive to Epo treatment than MMECs, probably because over-angiogenic phenotype of MMECs is already activated by their autocrine/paracrine loops occurring in the "angiogenic switch" from MGUS.

Abstract Purpose: The aim of this study was to investigate the angiogenic role of the hepatocyte growth factor (HGF)/cMET pathway and its inhibition in bone marrow endothelial cells (EC) from patients with multiple myeloma versus from patients with monoclonal gammopathy of undetermined significance (MGUS) or benign anemia (control group). Experimental Design: The HGF/cMET pathway was evaluated in ECs from patients with multiple myeloma (multiple myeloma ECs) at diagnosis, at relapse after bortezomib- or lenalidomide-based therapies, or on refractory phase to these drugs; in ECs from patients with MGUS (MGECs); and in those patients from the control group. The effects of a selective cMET tyrosine kinase inhibitor (SU11274) on multiple myeloma ECs' angiogenic activities were studied in vitro and in vivo. Results: Multiple myeloma ECs express more HGF, cMET, and activated cMET (phospho (p)-cMET) at both RNA and protein levels versus MGECs and control ECs. Multiple myeloma ECs are able to maintain the HGF/cMET pathway activation in absence of external stimulation, whereas treatment with anti-HGF and anti-cMET neutralizing antibodies (Ab) is able to inhibit cMET activation. The cMET pathway regulates several multiple myeloma EC activities, including chemotaxis, motility, adhesion, spreading, and whole angiogenesis. Its inhibition by SU11274 impairs these activities in a statistically significant fashion when combined with bortezomib or lenalidomide, both in vitro and in vivo. Conclusions: An autocrine HGF/cMET loop sustains multiple myeloma angiogenesis and represents an appealing new target to potentiate the antiangiogenic management of patients with multiple myeloma. Clin Cancer Res; 20(22); 5796–807. ©2014 AACR.

Abstract Purpose: The aim of the study was to verify the hypothesis that the cMet oncogene is implicated in chemio- and novel drug resistance in multiple myeloma. Experimental Design: We have evaluated the expression levels of cMET/phospho-cMET (p-cMET) and the activity of the novel selective p-cMET inhibitor (SU11274) in multiple myeloma cells, either sensitive (RPMI-8226 and MM.1S) or resistant (R5 and MM.1R) to anti–multiple myeloma drugs, in primary plasma cells and in multiple myeloma xenograft models. Results: We found that resistant R5 and MM.1R cells presented with higher cMET phosphorylation, thus leading to constitutive activation of cMET-dependent signaling pathways. R5 cells exhibited a higher susceptibility to the SU11274 inhibitory effects on viability, proliferation, chemotaxis, adhesion, and to its apoptogenic effects. SU11274 was able to revert drug resistance in R5 cells. R5 but not RPMI-8226 cells displayed cMET-dependent activation of mitogen-activated protein kinase pathway. The cMET and p-cMET expression was higher on plasma cells from patients with multiple myeloma at relapse or on drug resistance than on those from patients at diagnosis, complete/partial remission, or from patients with monoclonal gammopathy of unknown significance. Viability, chemotaxis, adhesion to fibronectin or paired bone marrow stromal cells of plasma cells from relapsed or resistant patients was markedly inhibited by SU11274. Importantly, SU11274 showed higher therapeutic activity in R5- than in RPMI-8226–induced plasmocytomas. In R5 tumors, it caused apoptosis and necrosis and reverted bortezomib resistance. Conclusion: Our findings suggest that the cMET pathway is constitutively activated in relapsed and resistant multiple myeloma where it may also be responsible for induction of drug resistance, thus providing the preclinical rationale for targeting cMET in patients with relapsed/refractory multiple myeloma. Clin Cancer Res; 19(16); 4371–82. ©2013 AACR.

Event Abstract Back to Event Bone marrow endothelial cells of patients with multiple myeloma: antigen presenting functions and T cell regulation ARIANNA FERRUCCI1, PATRIZIA LEONE1, ROBERTO RIA1, VITO RACANELLI1 and ANGELO VACCA1* 1 DEPARTMENT OF BIOMEDICAL SCIENCES AND HUMAN ONCOLGY - SECTION OF INTERNAL MEDICINE AND CLINICAL ONCOLOGY, Italy Immunity against cancer is hampered by local mechanisms promoting tumor evasion from T cell surveillance or tumor-specific T cell tolerance in the tumor microenvironment. We have analyzed the antigen presenting capacities of endothelial cells from bone marrow of patients with multiple myeloma (MM), an inexorably lethal plasma cell malignancy1. We have found that bone marrow endothelial cells basally express both class I and class II MHC molecules as well as the costimulatory molecules CD80 and CD86. The expression level of these molecules is slightly lower than that of professional antigen presenting cells, such as dendritic cells, from the same patients. Bone marrow endothelial cells also express the components of the MHC class I antigen processing machinery which include the constitutive proteasome subunits delta (beta1), MB1 (beta5) and zeta (beta2); the interferon-gamma (INF-gamma)-inducible proteasome (immunoproteasome) subunits LMP2 (beta1i), LMP7 (beta5i) and LMP10 (beta2i); the peptide transporters TAP1 and TAP2 and the endoplasmic reticulum chaperones calnexin, calreticulin, ERp57 and tapasin. When bone marrow endothelial cells are cultured, CD80 and CD86 expression decreases, class II MHC molecule largely disappears and class I MHC molecule does not significantly change2. Both class II MHC and costimulatory molecule expression are restored by bone marrow endothelial cell exposure to IFN-gamma and IL-6. Cultured bone marrow endothelial cells can activate purified allogeneic and autologous CD4 and CD8 T cells. Acknowledgements This work was supported by Associazione Italiana per la Ricerca sul Cancro (Italian Association for Cancer Research - AIRC), Investigator Grant and the 5 per thousand Molecular Clinical Oncology Special Program (grant n. 9965), Milan, to AV, the European Commission's Seventh Framework programme (EU FPT7) under grant agreement n° 278706 (OVER-MyR) to AV, and grants from MIUR PRIN 2009WCNS5C_004 to RR, and 2010NECHBX to AV. References 1. Vacca A, Ria R, Semeraro F, Merchionne F, Coluccia M, Boccarelli A et al. (2003). Endothelial cells in the bone marrow of patients with multiple myeloma. Blood 102: 3340–3348. 2. Pober JS and Tellides G (2012). Participation of blood vessel cells in human adaptive immune responses. Trends in Immunology 33, 1: 49–57. Keywords: Multiple Myeloma, Bone Marrow, Endothelial Cells, antigen presenting cells, T cells Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Adaptive Immunity Citation: FERRUCCI A, LEONE P, RIA R, RACANELLI V and VACCA A (2013). Bone marrow endothelial cells of patients with multiple myeloma: antigen presenting functions and T cell regulation. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00931 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Prof. ANGELO VACCA, DEPARTMENT OF BIOMEDICAL SCIENCES AND HUMAN ONCOLGY - SECTION OF INTERNAL MEDICINE AND CLINICAL ONCOLOGY, BARI, Italy, angelo.vacca@uniba.it Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers ARIANNA FERRUCCI PATRIZIA LEONE ROBERTO RIA VITO RACANELLI ANGELO VACCA Google ARIANNA FERRUCCI PATRIZIA LEONE ROBERTO RIA VITO RACANELLI ANGELO VACCA Google Scholar ARIANNA FERRUCCI PATRIZIA LEONE ROBERTO RIA VITO RACANELLI ANGELO VACCA PubMed ARIANNA FERRUCCI PATRIZIA LEONE ROBERTO RIA VITO RACANELLI ANGELO VACCA Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Multiple myeloma plasma cells home and expand in the bone marrow where cause an unbalanced bone remodelling with increased bone resorption and low bone formation that represent the typical feature in the majority of patients. A clinically relevant aspect of the interactions of multiple myeloma plasma cells in the bone marrow microenvironment is neovascularization, a constant hallmark of disease progression. This process is only partially supported by factors such as vascular endothelial growth factor, fibroblast growth factor-2 and metalloproteinases, which are directly secreted by the tumor cells. In fact, the presence in the bone marrow microenvironment of cytokines, in particular interleukin-6, as a consequence of plasma cell-stromal cell interactions, induces the production and secretion of angiogenic factors by other cells present in the bone microenvironment, thus contributing to the angiogenic switch during the progression of the disease. Near angiogenesis vasculogenesis occur in the bone marrow of myeloma patients and contribute to the vascular three formation. In the bone marrow of myeloma patients haematopoietic stem cells are recruited and induced to differentiate into endothelial cells by the angiogenic cytokines present in the microenvironment. Myeloma plasma cells also induce angiogenesis indirectly via recruitment and activation of stromal inflammatory cells (i.e.: macrophages and mast cells) to secrete their own angiogenic factors. They are recruited and activated by tumor plasma cells through the secretion of fibroblast growth factor-2, interleukin-8, and other chemokines, such as ITAC, Mig, IP-10. When macrophages and mast cells are activated they secrete their angiogenic factors: fibroblast growth factor-2, vascular endothelial growth factor, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, which contribute to enhance the tumor neovascularization. Finally, myeloma macrophages when exposed to vascular endothelial growth factor and fibroblast growth factor-2 secreted by plasma cells shows vasculogenic ability and acquire endothelial cell markers and transform into cells functionally and phenotypically similar to paired bone marrow endothelial cells. So they participate to the formation of the bone marrow capillary network (vasculogenic mimicry).

AbstractPurpose: To determine the in vivo and in vitro antiangiogenic power of lenalidomide, a “lead compound” of IMiD immunomodulatory drugs in bone marrow (BM) endothelial cells (EC) of patients with multiple myeloma (MM) in active phase (MMEC).Experimental Design: The antiangiogenic effect in vivo was studied using the chorioallantoic membrane (CAM) assay. Functional studies in vitro (angiogenesis, “wound” healing and chemotaxis, cell viability, adhesion, and apoptosis) were conducted in both primary MMECs and ECs of patients with monoclonal gammopathies (MGUS) of undetermined significance (MGEC) or healthy human umbilical vein endothelial cells (HUVEC). Real-time reverse transcriptase PCR, Western blotting, and differential proteomic analysis were used to correlate morphologic and biological EC features with the lenalidomide effects at the gene and protein levels.Results: Lenalidomide exerted a relevant antiangiogenic effect in vivo at 1.75 μmol/L, a dose reached in interstitial fluids of patients treated with 25 mg/d. In vitro, lenalidomide inhibited angiogenesis and migration of MMECs, but not of MGECs or control HUVECs, and had no effect on MMEC viability, apoptosis, or fibronectin- and vitronectin-mediated adhesion. Lenalidomide-treated MMECs showed changes in VEGF/VEGFR2 signaling pathway and several proteins controlling EC motility, cytoskeleton remodeling, and energy metabolism pathways.Conclusions: This study provides information on the molecular mechanisms associated with the antimigratory and antiangiogenic effects of lenalidomide in primary MMECs, thus giving new avenues for effective endothelium-targeted therapies in MM. Clin Cancer Res; 17(7); 1935–46. ©2011 AACR.