The role of cancer-associated fibroblasts (CAFs) has not been previously studied in multiple myeloma (MM). Here, cytofluorimetric analysis revealed higher proportions of bone marrow (BM) CAFs in patients with active MM (both at diagnosis and relapse) compared with patients in remission or those with monoclonal gammopathy of undetermined significance or deficiency anemia (controls). CAFs from MM patients produced increased levels of transforming growth factor-β, interleukin-6, stromal cell-derived factor-1α, insulin-like growth factor-1, vascular endothelial growth factor and fibroblast growth factor-2 and displayed an activated and heterogeneous phenotype, which supported their origin from resident fibroblasts, endothelial cells and hematopoietic stem and progenitor cells via the endothelial-mesenchymal transition as well as mesenchymal stem cells via the mesenchymal transition, as both of these processes are induced by MM cells and CAFs. Active MM CAFs fostered chemotaxis, adhesion, proliferation and apoptosis resistance in MM cells through cytokine signals and cell-to-cell contact, which were inhibited by blocking CXCR4, several integrins and fibronectin. MM cells also induced the CAFs proliferation. In syngeneic 5T33MM and xenograft mouse models, MM cells induced the expansion of CAFs, which, in turn, promoted MM initiation and progression as well as angiogenesis. In BM biopsies from patients and mice, nests of CAFs were found in close contact with MM cells, suggesting a supportive niche. Therefore, the targeting of CAFs in MM patients may be envisaged as a novel therapeutic strategy.

Purpose: To determine a "gene/molecular fingerprint" of multiple myeloma endothelial cells and identify vascular mechanisms governing the malignant progression from quiescent monoclonal gammopathy of undetermined significance. Experimental Design: Comparative gene expression profiling of multiple myeloma endothelial cells and monoclonal gammopathy of undetermined significance endothelial cells with the Affymetrix U133A Arrays was carried out in patients at diagnosis; expression and function of selective vascular markers was validated by real-time reverse transcriptase-PCR, Western blot, and small interfering RNA analyses. Results: Twenty-two genes were found differentially expressed (14 down-regulated and eight up-regulated) at relatively high stringency in multiple myeloma endothelial cells compared with monoclonal gammopathy of undetermined significance endothelial cells. Functional annotation revealed a role of these genes in the regulation of extracellular matrix formation and bone remodeling, cell adhesion, chemotaxis, angiogenesis, resistance to apoptosis, and cell-cycle regulation. Validation was focused on six genes (DIRAS3, SERPINF1, SRPX, BNIP3, IER3, and SEPW1) not previously found to be functionally correlated to the overangiogenic phenotype of multiple myeloma endothelial cells in active disease. The small interfering RNA knockdown of BNIP3, IER3, and SEPW1 genes affected critical multiple myeloma endothelial cell functions correlated with the overangiogenic phenotype. Conclusions: The distinct endothelial cell gene expression profiles and vascular phenotypes detected in this study may influence remodeling of the bone marrow microenvironment in patients with active multiple myeloma. A better understanding of the linkage between plasma cells and endothelial cells in multiple myeloma could contribute to the molecular classification of the disease and thus pinpoint selective gene targets for more effective antiangiogenic treatments. (Clin Cancer Res 2009;15(17):5369-78) The unique markers expressed by tumor vasculature distinguish it from normal endothelium. These abnormalities reflect the pathologic nature of its induction and attempts to discover tumor endothelial cell markers have always been hampered by technical difficulties in isolating functionally intact and phenotypically stable endothelial cells from patient samples. St. Croix et al. (1) were the first to show that colorectal cancer endothelial cells overexpress specific transcripts as a result of qualitative

Postzygotic mutations of the PIK3CA [phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha] gene constitutively activate the PI3K/AKT/mTOR pathway in PIK3CA-related overgrowth spectrum (PROS) patients, causing congenital mosaic tissue overgrowth that even multiple surgeries cannot solve. mTOR inhibitors are empirically tested and given for compassionate use in these patients. PROS patients could be ideal candidates for enrolment in trials with PI3K/AKT pathway inhibitors, considering the “clean” cellular setting in which a unique driver, a PIK3CA mutation, is present. We aimed to assess the effects of blocking the upstream pathway of mTOR on PROS patient-derived cells by using ARQ 092, a potent, selective, allosteric, and experimental orally bioavailable and highly selective AKT-inhibitor with activity and long-term tolerability, currently under clinical development for treatment of cancer and Proteus syndrome. Cell samples (i.e., primary fibroblasts) were derived from cultured tissues obtained from six PROS patients [3 boys, 3 girls; aged 2 to 17 years] whose spectrum of PIK3A-related overgrowth included HHML [hemihyperplasia multiple lipomatosis; n = 1], CLOVES [congenital lipomatosis, overgrowth, vascular malformations, epidermal nevi, spinal/skeletal anomalies, scoliosis; n = 1], and MCAP [megalencephaly capillary malformation syndrome; n = 4]. We performed the following: (a) a deep sequencing assay of PI3K/AKT pathway genes in the six PROS patients’ derived cells to identify the causative mutations and (b) a pathway analysis to assess the phosphorylation status of AKT [Ser473 and Thr308] and its downstream targets [pAKTS1 (Thr246), pRPS6 (Ser235/236), and pRPS6Kβ1 (Ser371)]. The anti-proliferative effect of ARQ 092 was tested and compared to other PI3K/AKT/mTOR inhibitors [i.e., wortmannin, LY249002, and rapamycin] in the six PROS patient-derived cells. Using ARQ 092 to target AKT, a critical node connecting PI3K and mTOR pathways, we observed the following: (1) strong anti-proliferative activity [ARQ 092 at 0.5, 1, and 2.5 μM blunted phosphorylation of AKT and its downstream targets (in the presence or absence of serum) and inhibited proliferation after 72 h; rapamycin at 100 nM did not decrease AKT phosphorylation] and (2) less cytotoxicity as compared to rapamycin and wortmannin. We demonstrated the following: (a) that PROS cells are dependent on AKT; (b) the advantage of inhibiting the pathway immediately downstream of PI3K to circumventing problems depending on multiple classes a PI3K kinases; and (c) that PROS patients benefit from inhibition of AKT rather than mTOR. Clinical development of ARQ 092 in PROS patients is on going in these patients.Electronic supplementary materialThe online version of this article (10.1007/s10048-018-0540-1) contains supplementary material, which is available to authorized users.

Purpose: 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 mmol/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.