ALBERT

Description: Angiogenesis has a critical role in the pathophysiology of multiple myeloma (MM); however, the molecular mechanisms underlying this process are not completely elucidated. The new tumor-suppressor gene inhibitor of growth family member 4 (ING4) has been recently implicated in solid tumors as a repressor of angiogenesis. In this study, we found that ING4 expression in MM cells was correlated with the expression of the proangiogenic molecules interleukin-8 (IL-8) and osteopontin (OPN). Moreover, we demonstrate that ING4 suppression in MM cells up-regulated IL-8 and OPN, increasing the hypoxia inducible factor-1α (HIF-1α) activity and its target gene NIP-3 expression in hypoxic condition. In turn, we show that the inhibition of HIF-1α by siRNA suppressed IL-8 and OPN production by MM cells under hypoxia. A direct interaction between ING4 and the HIF prolyl hydroxylase 2 (HPH-2) was also demonstrated. Finally, we show that ING4 suppression in MM cells significantly increased vessel formation in vitro, blunted by blocking IL-8 or OPN. These in vitro observations were confirmed in vivo by finding that MM patients with high IL-8 production and microvascular density (MVD) have significantly lower ING4 levels compared with those with low IL-8 and MVD. Our data indicate that ING4 exerts an inhibitory effect on the production of proangiogenic molecules and consequently on MM-induced angiogenesis.

Description: Osteoblast suppression is the hallmark of Multiple Myeloma (MM) osteolytic bone lesions mainly due to the capacity of MM cells to inhibit the osteogenic differentiation of bone marrow (BM) mesenchymal cells (MSC). Many evidences suggest that Wnt signaling is critically involved in the regulation of osteoblast formation. Recently, in murine osteoprogenitor cells and in MM mouse models it has been shown that activation of canonical Wnt pathway stimulate osteoblast formation and blunts MM-induced bone destruction. In this study we have investigated whether modulation of both canonical and noncanonical Wnt signaling pathway may affect osteogenic differentiation of human MSC and counterbalance the suppressive effect of MM cells. First we checked the potential expression of Wnt activators and inhibitors by human MSC and osteoprogenitor cells (PreOB) by gene arrays. We found that both cells expressed the activator of non-canonical Wnt pathways Wnt5a but lack of express the main activators of canonical Wnt signaling as Wnt1, Wnt3a and Wnt8. The presence of the Wnt5a receptor FZD2 and FZD5 was also detected in both cells as well as that of FZD3, FZD6 and FDZ7 and the Wnt canonical co-receptors LRP5 and LRP6. On the other hand we found that both inhibitors of canonical and non-canonical Wnt pathways DKK-1 and sFRP-1 were expressed by MSC. Secondly, activation of either canonical or non-canonical Wnt signaling pathway by Wnt3a and Wnt5a treatment respectively was performed in human MSC to evaluate the effect on osteogenic differentiation and the expression of osteoblast related markers (Collagen I, Osteocalcin and Alkaline Phosphatase). We found that Wnt5a treatment but not Wnt3a significantly increased the early osteogenic differentiation and the expression of alkaline phosphatase in MSC. Consistently in a co-culture system with MM cells Wnt5a treatment blunted, at least in part, the inhibitory effect of MM cells on alkaline phosphatase expression by MSC and PreOB. To go further inside, we evaluated in both primary human BM MSC and the human MSC cell line HS-5 the effect of either the activation of non-canonical Wnt signaling by Wnt5a overexpression using a lentivirus vector or the Wnt5a suppression using siRNA. Wnt5a over-expression in MSC induced the activation of Wnt/Ca++ non-canonical pathway as demonstrated by the increase of Wnt5a secretion and phospho-PKC expression detected by westernblot analysis. Consequently to non-canonical Wnt signal activation we found a significant increase of alkaline phosphatase expression by MSC cells as well as of their osteogenic differentiation. Interestingly, analyzing the gene expression profile by microarray, we found that Wnt5a overexpression in MSC also affects the expression of chemokines, inflammatory cytokines and pro-angiogenic molecules. In conclusion our data indicate that activation of non-canonical Wnt signal pathway may represent a potential target in MM microenvironment to counterbalance the inhibitory effect of MM cells on osteogenic differentiation of human MSC.

Description: Multiple Myeloma (MM) cells are extremely resistant to apoptosis and currently new potential drug combinations are under investigation. We have shown that the combined treatment with the MEK1/2 inhibitor PD184352 (PD) and Arsenic Trioxide (ATO) resulted in the synergistic (Combination Index

Description: Abstract 1482 Poster Board I-505 Alterations in cell trafficking and chemoattractive properties may represent basic mechanisms responsible for organ pathology in diabetes mellitus (DM). These functional events involving both inflammatory and progenitor cells may be translated in the proneness to major septic events and effaced reparative response characterizing the clinical course of the disease. To determine whether DM affects BM progenitor cell number and function, tissue resident and GSF mobilized disease-free BM CD34pos cells were evaluated in 19 DM and 38 non-diabetic control (CTR) biopsies and in 18 DM and 48 CTR patients undergoing autologous BM transplantation. DM was documented by the presence of glucose intolerance, HbA1C 〉 6 g% and need of high dose insulin treatment. CTR were selected on the basis of normal glucose tolerance and the absence of neoplastic infiltration and/or morphologic changes in cellular hematopoietic compartments compatible with hematologic disorders. Reduction in trabecular bone, fat accumulation, rearrangement of microvascular structures and marked DNA oxidative damage characterized BM structural and cellular remodelling in DM. Immunohistochemical and FACS analysis documented that DM produced a 50% reduction in the number of both BM resident and mobilized CD34pos Linneg cells. In addition only 16% of diabetic patients were able to mobilize 〉10 × 106 CD34pos cells/kg compared to 62% of CTR. After infusion of mobilized CD34pos progenitors, BM engraftment was partially prolonged in diabetic patients. Severe impairment of SC engraftment by DM was striking by the comparative analysis of the infusion of similar number of mononuclear cells (nearly 180 ×106/Kg) directly harvested from the bone marrow of DM and CTR patients. In this setting, PMN and Plts recovery was delayed by nearly 2 and 4 weeks, respectively. Thus, DM severely affects human progenitor cell function and BM microenvronment in vivo inevitably impairing the outcome of BM transplantation. Whether all these changes are due to specific perturbation of the GSF-dependent SDF-1/CXCR4 axis and whether drugs interfering with ROS mediated damage may reverse this phenomenon is under intense investigation. Disclosures: No relevant conflicts of interest to declare.

Description: Gene expression alterations occurring in the bone microenvironment cells and their potential relationships with the occurrence of bone lesions in multiple myeloma (MM) patients have never been investigated. In this study, we have isolated both mesenchymal (MSC) and osteoblastic (OB) cells, without in vitro differentiation, from bone biopsies obtained by iliac crest of 24 MM patients, 7 MGUS subjects and 8 healthy donors (N) who underwent orthopedics surgery. Bone status was evaluated in all MM patients by total X rays scan and MRI for the spine. Firstly, we evaluated cell proliferation in relationship with growth substrate (bone and glass) and cell phenotype by flow cytometry and immunohistochemistry. We found that both MSC and OB cells have higher cell doubling rate in MM patients as compared to N. Higher expression of alkaline phosphatase and Runx2 was observed in OB as compared to MSC cells in both N and MM patients without osteolytic lesions, but not in osteolytic ones. We performed a gene expression profiling analysis of isolated MSC and OB cells using GeneChip® Affymetrix HG-U133A oligonucleotide arrays. An unsupervised analysis of the most variable genes across the dataset generated a hierarchical clustering with the two major branches containing respectively MSC and OB samples. A multiclass analysis of N, MGUS and MM patients identified 33 differentially expressed probe-set (specific for 27 genes) in MSC cells, and 19 differentially expressed probe-set (13 genes) in OB, and the identified transcripts mainly characterized N versus MM and MGUS samples. A supervised analysis between N and MM samples identified 65 probes (56 genes: 17 up-regulated and 39 down-regulated) differentially expressed in MSC and 35 probes (29 genes, 12 up-regulated and 17 down-regulated) in OB. Notably, genes encoding the Homeobox class proteins, such as HOXB2-6-7, were up-regulated in both MSC and OB of MM patients as compared to N. As regards the bone status, a total of 60 probe-sets (3 up-regulated and 57 down-regulated genes) were found differentially expressed in MSC from osteolytic vs. non-osteolytic MM patients, whereas MGUS-MSC exhibited an intermediate transcriptional profile between osteolytic and non-osteolytic MM patients. A distinct pattern of gene expression profiling was also observed in MSC versus OB when osteolytic and non-osteolytic MM patients were compared (26 vs. 94 differentially expressed probe-sets, respectively), including transcription factors related to MSC osteogenic differentiation belonging to Runx2 pathway (HEY1) or Wnt and BMP signaling On the other hand, few genes were found differentially expressed in OB cells in relationship with the presence of bone lesions. In conclusion, we identified a distinctive transcriptional fingerprint in isolated MSC and OB cells of MM patients as compared to N subjects, which mainly correlated with cell proliferation. Moreover, a different gene expression profile was observed in MSC cells of MM patients according to the presence/absence of bone lesions, highlighting the critical role of the block of the osteogenic differentiation.

Description: Bone marrow (BM) neo-angiogenesis has a critical role in multiple myeloma (MM) progression. It is well established that the angiogenic process in MM is mainly due to an overproduction of pro-angiogenic molecules by MM cells and the BM microenvironment cells. However the molecular mechanisms at the basis of the angiogenic process in MM are currently under investigation. The deregulation of the homeobox genes has been previously associated to tumor progression and neoangiogenesis. Particularly, overexpression of the homeobox HOXB7 is critical in tumor-associated angiogenic switch in solid tumors as breast cancer. Actually the potential role of HOXB7 in MM-induced angiogenesis is not known. In this study we have investigated the expression of HOXB7 by MM and BM microenvironment cells and its potential role in the regulation of the angiogenic process. First, by microarray analysis in a large database of MM patients (n°= 132) we found that HOXB7 was overexpressed by MM cells in about 10% of patients as compared to healthy donors and MGUS subjects. On the other hand HOXB7 mRNA was expressed in 18 out of 23 human myeloma cell lines tested. Moreover, we found that isolated BM mesenchymal (MSC) and osteoblastic (OB) cells, obtained from bone biopsies in a subgroup of MM patients (n°=24) expressed HOXB7 gene by microarray analysis and real time PCR. HOXB7 expression was also investigated at protein level by immunohistochemistry on bone biopsies of MM patients finding that MSC and OB as well as endothelial cells expressed HOXB7 protein mainly at nuclear level. In order to investigate the potential role of HOXB7 in the angiogenic process we enforced HOXB7 expression by lentivirus vectors in MSC using both primary BM MSC and the human MSC cell line HS-5 to obtain a stable transduced cell line. The overexpression of HOXB7 in HOXB7 transduced MSC as compared to the empty vector-transduced MSC cells was confirmed by real time PCR, western blot and immunohistochemistry. By Gene chips U133 plus 2.0 (Affymetrix) we evaluated the gene expression profiling of HOXB7 over-expressing MSC finding that proangiogenic cytokines, metalloproteinases and chemokines were significantly modulated in HOXB7-transduced MSC cells as compared to control cells. Data were validated either by real time PCR or by western blot and by an angiogenesis antibody array showing that bFGF and VEGF production was induced in MSC by HOXB7 overexpression. Consistently, we found that conditioned media of HOXB7-transduced MSC cells significantly stimulated vessel formation as compared to controls using an in vitro angiogenic model. Finally we observed that the angiogenic in vitro differentiation of HOXB7-transduced MSC was significantly increased as compared to controls. In conclusion our data suggest the HOXB7 overexpression in MSC regulates the angiogenic switch and could be a potential therapeutic target in MM-induced angiogenesis.

Description: Hypoxia is a common feature of solid tumors associated to angiogenesis and malignant phenotype. Tumor adaptation to hypoxia is mainly due to the hypoxia-inducible factor (HIF)-1α, a key transcription factor that regulates angiogenesis and tumor progression. As known multiple myeloma (MM) is a hematological malignancy characterized by the accumulation of malignant plasmacells into a hypoxic microenvironment as the bone marrow (BM) that critically supports their growth and survival. However the effect of hypoxia on MM cells and the role of HIF-1α in MM-induced angiogenesis actually are not known. Recently we have demonstrated that the tumor suppressor gene ING4 may exert an anti-angiogenic effect through the inhibition of HIF-1α activity in MM cells in hypoxic condition suggesting a role of HIF-1α in MM-induced angiogenic switch. To go further insight this issue, in this study, first we checked the level of BM oxygen tension in a cohort of MM patients (n°=25) at the diagnosis as compared to healthy donors and MGUS subjects. The mean pO2 ± SD was 52.3±9 mmHg (p=NS) in MM patients similar to that observed in the controls, confirming that MM cells are exposed in vivo to hypoxic microenvironment. Thereafter HIF-1α protein expression by MM cells was checked by immunohistochemistry on bone biopsies showing the presence of HIF-1α stabilization at nuclear level in malignant plasmacells as well as in BM stromal cells (BMSC) into the BM. Consequently the effect of hypoxia and HIF-1α in both MM and BMSC cells was checked. Human myeloma cell lines (JJN3 and RPMI-8226) and BMSC were transfected with a pool of siRNA anti-HIF-1α to knockout HIF-1α and then exposed to low oxygen tension. A gene expression profiling evaluation was performed by microarray analysis using Gene Chips U133plus 2.0 (Affymetrix). Data were then validated by real time PCR. We found that hypoxia significantly upregulated the expression of the pro-angiogenic molecules in both MM and BMSC cells including Vascular Endothelial Growth Factor (VEGF), Osteopontin (OPN) and Interleukin-8 (IL-8) blunted by siRNA anti-HIF-1α. Genes belonging to glycolysis and HIF-1α regulating signal pathways were found to be also regulated by HIF-1α in MM cells in hypoxic condition. These observations were confirmed in purified CD138+ MM cells (n°=11) exposed to hypoxia that induced a significant up-regulation of the pro-angiogenic molecules and the modulation of glycolysis and ubiquitin mediated proteolysis signal pathways. Finally, the potential expression and role of HIF-1α in MM cells was also investigated in normoxic condition. Whereas the presence of HIF-1α mRNA was observed in all HMCLs and primary MM cells tested, HIF-1a protein stabilization and activity was observed at nuclear level in 2 out of 6 HMCLs and in about 38% of MM patients evaluated suggesting that a hypoxia independent stabilization of HIF-1α may occur in MM cells. Consistently, in normoxic condition, HIF-1α knock out by siRNA significantly affected in HMCLs either pro-angiogenic molecules as VEGF or several genes belonging to cell cycle regulation. In conclusion our data underline the role of hypoxia in the regulation of the angiogenic signature of MM cells and the BM microenvironment and suggest that HIF-1α could be a potential target in MM.

Description: Abstract 334 Background: Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease believed to be mediated by autoreactive lymphocytes that invade the Central Nervous System and cause oligodendrocyte, axonal and neuronal damage as well as glial scarring.to and resulting in demyelination, neuronal death and brain atrophy. Hematopoietic Stem Cell Transplantation (HSCT) has been tried in the last 15 years as a therapeutic option in patients with a poor prognosis autoimmune disease not responding to conventional treatments. Worldwide ≥600 patients with MS have been treated with HSCT, most of them having been recruited in small, single center, phase 1–2 uncontrolled trials. Clinical and MRI outcomes from case series reports or Registry-based analyses suggest that a major response is achieved in most patients; quality and duration of response are better in patients transplanted earlier in the relapsing-remitting phase. The intensity of the immunosuppression from transplant treatment may be determined by several factors, such as the use of chemotherapy in the mobilization regimen, the intensity of the conditioning regimen and ex-vivo T-cell depletion. Methods: We report here the Italian multi-center experience on 74 MS patients treated with AHSCT between 1996 and 2008, all mobilized with Cyclophosphamide/G-CSF and conditioned with BEAM and rabbit ATG. Clinical and MRI outcomes were reported to the Italian Registry; the median follow-up is 48.3 (range 30–210) months. All patients clinically deteriorated in the year prior to HSCT, with an increase of Extended Disability Scale (EDSS) of at least one point (average EDSS change=1.5 points, range=1–9), Results: Two patients (3.3%) died for transplant-related causes. At 5 years after the transplant, 66% of patients remained stable or improved. Progression free survival (PFS) was slightly better in relapsing-remitting (RR) (5 years PFS=71%) than in secondary-progressive (SP) forms (5 years PFS=62%, p=0.28). Amongst patients with a follow up longer than 1 year, 8 out of 25 RR subjects (31%) had a 6–12 months confirmed EDSS improvement 〉 1 point as compared to 1 out of 36 (3%) SP patients (p=0.009), Figure 1. Out of 18 cases with a follow up longer than 7 years, 8 (44%) remained stable or had a sustained improvement whilst 10 (56%), after an initial period of stabilization or improvement with a median duration of 3.5 years, showed a slow progression of disability. Conclusions: This study shows in a large cohort of patients with a long follow-up that AHSCT with BEAM/ATG conditioning regimen has a profound effect in suppressing disease progression in aggressive MS cases, unresponsive to conventional therapies. Indeed it results in a sustained improvement of the disability, free of immunosuppression, in a significant amount of RR patients. Clinical improvement is scarcely reported in MS literature as it is rather infrequent with conventional treatments; nevertheless it results in a stable increase of the quality of the life in this subset of young patients and should be considered in the choice of a therapeutic strategy in the early phase of MS. Disclosures: Cuneo: Roche: Consultancy, Speakers Bureau.

Description: Bone marrow (BM) angiogenesis is increased in Multiple Myeloma (MM) patients and correlates with disease progression and patient survival. Myeloma cells secrete the main endothelial growth factor VEGF. In mouse models VEGF secretion as well as the angiogenic properties of MM cells correlate with the lack of CD45 expression by MM cells. However, recent data indicate that VEGF plasma cell expression is similar between MGUS and MM patients suggesting that other molecules could be involved. In line with this hypothesis we have recently demonstrated that myeloma cells may also produce factors with angiogenic properties as angiopoietin-1 (ANG-1) and osteopontin (OPN) that are involved in myeloma induced angiogenesis in vitro. In order to identify which factors correlate with BM angiogenesis in MM patients, we have investigated in a cohort of 121 newly diagnosed MM patients (stage I–III) the expression of the angiogenic molecules VEGF, ANG-1 and OPN and their correlation with bone marrow (BM) angiogenesis and CD45 expression by MM cells. We found that 90% of CD138+ MM cells tested were positive for VEGF mRNA. On the other hand we found that 50% and 40 % of MM patients were positive for ANG-1 and OPN mRNA respectively. Using the previously published cut off for CD45 expression we found that 61 out of 121 MM patients were positive for CD45 and 60 out of 121 were negative for CD45 expression. Any correlation was not observed between VEGF expression and BM angiogenesis in MM patients (p=0.5), whereas the number of microvessels X field was higher in Ang-1 positive patients in comparison with Ang-1 negative ones (mean±SE: 6.23±0.2 vs. 2.94±0.1, median: 6.21 vs. 2.79; p=0.001,) and the microvascular density (MVD) was significantly increased (32.98±1.7 vs. 14.55±1.3, median: 34.69 vs. 13.04; p