ALBERT

Description: Abstract 3016 Background: Multiple myeloma (MM) is an incurable disease characterized by genetically transformed clonal plasma cells that develop a proliferative advantage within the supportive bone marrow (BM) microenvironment. Recent findings from our and other laboratories have shown genetically unstable endothelial progenitor cells (EPCs) to be a key component of the MM microenvironment, integral to tumor neovascularization. However, the contribution and characterization of genomic alterations in the tumor microenvironment in the progression of MM has not been established. Using array-comparative genomic hybridization (aCGH), the present study examined genome-wide copy number alterations (CNAs) within the EPC genome and compared them to tumor cells and control endothelial cells (ECs). Published human copy number variations (CNVs) were excluded from the analyses. Methods: Informed consent was obtained from all subjects. EPCs (〉98% vWF/CD133/KDR+/CD38–) from BM aspirates of 16 untreated MM patients were outgrown on laminin-coated flasks. Controls included EPCs from healthy subjects and human umbilical vein ECs (HUVECs). For microarray analysis, genomic DNA from paired EPCs and tumor cells from MM patients enriched for CD38+ cells, as well as control cells, were hybridized to Agilent 244A Human Genome CGH Microarrays with differentially labeled control peripheral blood mononuclear cells, and feature intensities and ratios were extracted in Agilent CGH Analytics Software. The aberration detection method-1 algorithm was used to assess intervals in which the average log2 ratio of the MM cells and EPCs to control cells and ECs exceeded 0.3 (at least 1.23 fold-change). Human Genome Structural Variation Project (humanparalogy.gs.washington.edu) and the Database of Genomic Variants (projects.tcag.ca/variation) served as control CNVs. Affymetrix U133 plus 2.0 GeneChips confirmed gene expression using GeneSpring software (Agilent), and group comparisons were made by ANOVA. Results: Extensive chromosomal CNAs were found in MM EPCs; gains and losses were found to approximately the same extent in matched tumor cells. Germline CNVs accounted for less than 10% of MM EPC CNAs. The greatest number of CNA gains in EPCs were found on chr 7q, followed by 2p and 22q; the most recurrent sequences with CNA gains were on chr 7. The greatest CNAs losses in MM EPCs were found equivalently on chr 1q, 11q, and 15q. Consistent with their clonal expression in MM, immunoglobulin genes were found to be dysregulated in MM EPCs (e.g., 14q32 gains), which were confirmed at the gene expression level (e.g., over-expression of IGHG1 mRNA compared to control ECs). When comparing CNAs in MM EPCs to those in corresponding tumor cells, 15 of 16 patients (94%) shared identical CNAs at 2 or more loci, with greater than 48% similarity in CNAs between EPCs and tumor cells. Control EPCs and HUVECs did not show significant baseline alterations compared to control normal lymphocyte DNA, whereas identical CNAs were found in MM EPCs and tumor cells throughout their genomes. The most recurrent CNAs in both EPCs and tumors were found on chr 1 and 14, which are known to be highly dysregulated in MM. The clinical relevance of our aCGH data is suggested by the finding that more CNA gains and losses were found both in EPCs and in tumor cells from MM patients with treatment-resistant, progressive MM than in patients in remission (P

Description: Abstract 450 Background: Multiple myeloma (MM) is a disease of clonal plasma cells that accumulate in the bone marrow (BM), causing monoclonal immunoglobulin (IG) production, BM failure, osteolytic lesions, and kidney disease. Although initially treatable, tumor cells ultimately become resistant to drug-treatment, and the disease is invariably fatal. Therefore, novel treatment targets need to be identified. The tumor microenvironment, and vascular endothelial cells in particular, play a key role in the adhesion and migration of MM cells and thus govern tumor survival and growth, as well as the acquisition of drug-resistance. Hence, the adhesion/migration systems of MM cells are key potential therapeutic targets. The cell membrane protein JAM-A/F11R is an endothelial cell (EC) adhesion molecule of the IG superfamily, and its expression is upregulated by TNF-a through NF-κB signaling. F11R also alters EC migration and paracellular permeability via stabilization of β1 integrin. We have previously shown that F11R gene expression and serum levels are upregulated in patients with MM compared to healthy controls. In this study, we further explored the functions of F11R within MM cells in order to gain insight into the potential role of this molecule in the progression and treatment of MM. Methods: The MM cell line RPMI-8266 (RPMI) was examined for functional studies in vitro. Informed consent was obtained from all subjects. Primary BM tumor cells were enriched to 〉 95% CD138+ cells by positive selection using anti-CD138 MACS MicroBeads. The CD138– fraction was used for outgrowth of confluent EPCs (〉 98% vWF/CD133/KDR+). Human umbilical vein endothelial cells (HUVECs) served as controls. F11R mRNA levels were assessed by Affymetrix GeneChip analysis and by F11R probe-based real-time PCR compared to a standard curve normalized to GAPDH mRNA levels. F11R protein levels were measured by immunofluorescence (IF) and flow cytometry. The role of F11R in MM cell migration and survival was quantified by examining these functions in RPMI cells in which F11R was knocked down by siRNA silencing and comparing them with control untransfected RPMI cells or cells transfected with a non-targeting siRNA or lipofectamine. Tumor migration and survival were determined by the Millipore QCM Chemotaxis assay (using a 5 micron pore size) and an Promega Cell Proliferation Assay, respectively. Each assay was performed in triplicate and replicated at least twice. Statistical analyses were performed using Student's t-test, two-tailed; P≤.05 was considered significant. Results: Inhibition of F11R gene expression by siRNA resulted in 70% cell death compared to control untransfected (P

Description: Abstract 2830 Poster Board II-806 Background: Multiple myeloma (MM) is an incurable disease of clonal plasma cells that accumulate in the bone marrow (BM), causing monoclonal IG production, bone marrow failure, osteolytic lesions and kidney disease. Although initially treatable, MM ultimately becomes refractory to treatment, and is invariably fatal, when tumor cells that harbor genetic mutations expand without regulation. Therefore novel treatment targets need to be identified. A key mechanism in MM pathogenesis is regulation of tumor growth by the bone marrow (BM) microenvironment, particularly by bone marrow neo-vascularization and adhesion of tumor cells to the marrow stroma. Aberrantly expressed genes that regulate angiogenesis by MM cells enhance MM progression and constitute targets in its treatment. JAM-A/F11R is an endothelial cell (EC) adhesion molecule of the immunoglobulin superfamily which is a multifunctional cell membrane protein that mediates intracellular signaling events that alter EC migration and paracellular permeability. For example, in breast cancer, attenuation of JAM-A increases tumor invasion and metastasis through a decrease in tumor adhesion (Ulas Naik Cell Adh Migr. 2008 Oct;2(4):249-51.). In this study we explored the JAM-A/F11R expression in MM tumor cells and in patients to determine the potential role of this molecule in the pathogenesis and progression of MM. Methods: The MM cell lines examined were RPMI-8266, U266, and NCI-H929. Human umbilical vein endothelial cells (HUVECs) served as controls. Informed consent was obtained from patients and control subjects. Primary BM tumor cells were enriched to 〉 95% CD138+ cells by positive selection using anti-CD138 MACS MicroBeads. The CD138-negative fraction was used for outgrowth of confluent EPCs (〉 98% vWF/CD133/KDR+). JAM-A mRNA expression was assessed using an microarray gene expression profile, JAM-A probe based real-time PCR, and JAM-A levels in each sample were measure using a standard curve and normalized to GADPH. JAM-A protein levels in MM cell lines and primary tumor cells were measured by flow cytometry and immunofluorescence. For serum studies, peripheral blood was obtained from 25 newly diagnosed MM patients and 8 healthy, age- and sex-matched controls, and JAM-A levels were measured using an ELISA. Statistical analysis was performed using Student's t-test, two-tailed, with P ' .05 considered significant. Results: JAM-A mRNA levels were significantly increased in MM cell lines RPMI-8266, U266, and NCI-H929 compared to HUVECs (U266, P = 3×10-5; RPM1-8266, P = 1×10-6; NC1-H929, P= 5×10-4). The JAM-A mRNA levels were significantly greater in RPMI-8226; P 〈 .04 compared to TNFα-activated HUVECs for 24 hours which is a proangiogenic switch for HUVEC gene expression. The elevated mRNA expression of the JAM-A in MM cell lines was confirmed by immunofluorescence and flow cytometry which showed the presence of both membrane and cytoplasmic JAM-A protein. Microarray analysis of gene expression profiles from 20 patients' corresponding tumor cells and microenvironmental EPCs showed that JAM-A had a higher level of expression in tumor cells versus MM EPC by 12.62 fold, (P=.0000642). Furthermore, JAM-A had a higher level of expression in MM EPC versus normal control EPC by 2.41 fold, (P=.00113) reflecting a complex regulatory role of F11 signaling in MM, similar to breast cancer (Naik, U. et al 2008). JAM-A was also found to be 12.6 fold greater in tumor cells compared to EPCS (P=.0000642). In addition, circulating levels of soluble JAM-A were found to be significantly greater in the serum of MM patients compared to controls (P 〈 .005), with an average 2-fold increase. Serum levels of JAM-A in MM patients also decreased 71% with treatment n=5, P

Description: Abstract 2984 Background: Multiple myeloma (MM), a neoplasm of committed B-lymphocytes within the bone marrow (BM), is the second most common hematologic malignancy in the U.S. Despite prolonged median survival with anti-myeloma strategies aimed at the tumor and its BM microenvironment, MM remains invariably fatal. Endothelial progenitor cells (EPCs) are CD133+/KDR+ cells that originate in the BM and play a key role in supporting tumor growth and MM progression. Using X-chromosome inactivation and RT-PCR analyses, we previously found EPCs from MM patients to be clonally restricted and to display clonotypic IG heavy-chain gene rearrangements identical to the same patients' tumor cells (Braunstein et al., 2006). Based on the shared genetic identity that we and others have demonstrated between tumor cells and EPCs in MM patients, the present study explored the hypothesis that, similar to hemangioblasts, which are CD133-expressing precursors to adult hematopoietic and endothelial cells, EPCs may be a source of vascular and MM progenitor cells. Since hemangioblasts are known to exist predominately in the quiescent phases of the cell cycle, in this study we also examined the cell cycle status of CD133-expressing BM cells from MM patients in order to gain insight into their hemangioblastic traits. Methods: BM aspirates were acquired from MM patients under IRB approval. EPCs (〉98% vWF/CD133/KDR+ and CD38-) from BM aspirates of MM patients were outgrown on laminin-coated flasks as previously described. The spleen colony assay was used to determine the stem cell capacity within BM-derived EPCs by i.v. injection into NOD/SCID mice. The spleens and BM of mice were harvested 2–4 weeks later. Cells were analyzed by immunofluorescence (IF) and flow cytometry. Hoechst 33342 (Hst) and Pyronin Y (PY) were used to measure the cell cycle status of CD133+ cells using FACS analysis. Results: Two to four weeks following i.v. injection of MM EPCs, human cell surface marker expression detected by flow cytometry within mouse BM and spleen cells shifted from CD133+/CD45-lo, a progenitor cell phenotype, to CD133−/CD45-hi, a more differentiated phenotype, suggesting the ability of MM EPCs to differentiate in vivo. Cell cycle analysis of the CD133+ population in BM cells of MM patients showed that these cells were predominantly non-cycling. On average, 60.5% of CD133+ cells were found to be in the G0/G1 phase of the cell cycle, as indicated by low levels of IF staining with Hst and PY. Conclusions: CD133+ cells are strong candidates as precursors to MM tumor and vascular progenitor cells. As quiescent cells are non-dividing, they often escape cytotoxic effects of chemotherapy, resulting in relapse, and therefore, identification of these cells is critical. Ongoing studies include the engraftment of CD133+ cells in the subcutaneous NOD/SCID gamma xenotransplant model and their growth in response to anti-myeloma strategies; results of these studies will be discussed. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Multiple myeloma (MM) remains incurable, primarily due to the development of dose-limiting toxicity and/or resistance to previously effective drugs. A rational approach is to develop new strategies that are synergistic with existing agents. Recent research indicates that inhibition of the molecular chaperone heat shock protein (Hsp) 70 may facilitate resistance to inhibitors of cellular protein quality control such as bortezomib in MM. This is indicated by upregulation of Hsp70 expression after treatment of MM with inhibitors of the proteasome or Hsp90. Endothelial progenitor cells (EPCs) are bone marrow (BM)-derived hematopoietic precursor cells that augment tumor neovascularization and govern MM severity, suggesting that EPCs are a potential target for novel antimyeloma strategies. In this study, we examined the antimyeloma effects of MAL3-101, a member of a new class of inhibitors of Hsp 70, on MM cell lines and patient-derived MM cells and EPCs. We also determined the synergy between the antimyeloma effects of MAL3-101 and inhibition of the proteasome and Hsp90. Methods: MM cell lines (NCI-H929, RPMI-8266, and U266), BM-derived MM cells, EPCs from untreated patients, control PBMCs, and BM cells were treated with MAL3-101, the proteasome inhibitor MG-132, the Hsp90 inhibitor 17-AAG, or DMSO, alone and in combination. Cell survival and apoptosis were assessed by the MTS assay and Annexin V-PI staining, respectively. Cell cycle progression and immunoblots were performed by standard methods. Synergistic effects were evaluated by determining the combination index (CI) using CalcuSyn software. Results: H929 cells were most sensitive to MAL3-101, with peak cytotoxicity at 40 h (IC50 8.3 μM). In contrast, MAL3-51, a less potent Hsp70 modulator, was less effective. Furthermore, FACS analysis showed that exposure to 10 μM MAL3-101 caused a time-dependent increase in apoptosis and inhibition of cell cycle progression, indicated by a 3-fold increase in the sub-G0/G1 phase and a 2.5-fold decrease in the G2/M phase populations in H929 cells. Also, immunoblot analysis showed a time-dependent increase in the cleavage of caspase-3 and PARP by MAL3-101 in H929 cells, indicating induction of apoptosis. When H929 cells were exposed to a range of concentrations of MAL3-101 or MG-132 alone and in combination, we found that the IC50 for the agents in combination decreased to 0.008 μM. Notably, when examined alone, each compound was ineffective at the IC50 concentration of the combined compounds. The synergistic cytotoxic actions of MAL3-101 and MG-132 on H929 cells occurred over a 10-fold range of concentrations (0.01–0.1 μM) (CI 〈 1), and also resulted in synergistic apoptosis. Similarly, we found that the combination of MAL3-101 and 17-AAG also decreased the IC50 for 17-AAG, from 0.4 μM to 0.03 μM. These results are consistent with the prediction that the antimyeloma effects of Hsp90 inhibition, which causes upregulation of Hsp70 gene expression, would be potentiated by simultaneous inhibition of Hsp70. Synergistic antimyeloma effects of MAL3-101 and MG-132 were also observed as a decrease in viability of MM cells by 33% ± 8 (mean ± SD; P=.02) and 44% ± 10 (P=.07), by MAL3-101 and MG-132, respectively, but by 75% ± 4 (P=.001) when the agents were combined. In addition, combined MAL3-101 and MG-132 reduced viability of BM EPCs, by 10% ± 14 (P=.7) and 16% ± 17 (P=.3), respectively, but in combination by 60% ± 7 (P=.001). The specificity of MAL3-101’s effect on MM cells was indicated by a lack of cytotoxicity in control cells. When intracellular and secreted IGs were quantitated, we observed that the relative amount of IG secretion was highest in H929 cells, which also demonstrated the highest sensitivity to MAL3-101-induced cytotoxicity, indicating that the sensitivity of MM cells to Hsp70 inhibition most likely arises from the added cellular stress of producing and secreting monoclonal IGs; however, there was no evidence for an upregulation of an unfolded protein response in these cells by XBP-1 mRNA splicing. Conclusions: These results show for the first time that exposure to the Hsp70 inhibitor MAL3-101, a new Hsp70 modulator, sensitizes MM tumor and EPCs to proteasome and Hsp90 inhibition. These data support a preclinical rationale for inhibition of Hsp70 function, either alone or in combination with other agents, as a novel therapeutic strategy for MM.