ALBERT

Description: Nuclear paraspeckle assembly transcript 1 (NEAT1) is a long non-coding RNA (lncRNA) reported to be frequently deregulated in various types of cancers and neurodegenerative processes. NEAT1 is an indispensable structural component of paraspeckles (PSs), which are dynamic and membraneless nuclear bodies that affect different cellular functions, including stress response. Furthermore, increasing evidence supports the crucial role of NEAT1 and essential structural proteins of PSs (PSPs) in the regulation of the DNA damage repair (DDR) system. This review aims to provide an overview of the current knowledge on the involvement of NEAT1 and PSPs in DDR, which might strengthen the rationale underlying future NEAT1-based therapeutic options in tumor and neurodegenerative diseases.

Description: Galectin-1 (Gal-1) is a lectin, involved in several processes related to cancer, including immunosuppression, angiogenesis, hypoxia, and metastases. However, the expression profiles of Gal-1 and its pathophysiological role in multiple myeloma (MM) cell growth, in the relationship between MM cells and the bone marrow (BM) microenvironment and in the MM-induced angiogenesis are unknown and were investigated in this study. Firstly we evaluatedGal-1 expression by CD138+ cells of a dataset of 133 MM patients at diagnosis (GSE16122) and 23 human myeloma cell lines (HMCLs) (GSE6205) or on a proprietary? dataset of primary mesenchymal stromal cells (MSCs) and osteoblasts (OBs) of 16 MM and 4 MGUS. CD138+ cells and HMCLs were positive for LGALS1 with no statistically significant differences. LGALS1 mRNA expression was positively correlated with 154 genes and negatively with 109 genes including ERG1 and SPARC. MSCs cells showed a higher expression of LGALS1 compared to the OBs and MM-OBs showed a higher expression of LGALS1 mRNA than that obtained from healthy subjects. Gene expression profiling (GEP) data were then validated by Real-Time PCR and western blot in freshly purified primary CD138+ and BM MSCs samples as well as in 6 HMCLs and in both human MSC (HS-5 and hMSC-Tert) and osteoblastic cell lines (HOBIT and HOB-01). Moreover, immunohistochemistry analyses on bone biopsies obtained from 12 MM, 9 smoldering MM, 9 MGUS and 3 plasma cell leukemia samples revealed an high level of Gal-1 protein expression by MM cells, OBs and vessels in all the patients tested. Secondly, we evaluated whether Gal-1 expression was regulated by hypoxia and by Hypoxia Inducible Factor-1a (HIF-1a) checking the effect of hypoxic treatment (1% of O2) and HIF-1α inhibition by shRNA lentivirus. We found that Gal-1 was upregulated in HMCLs upon hypoxic treatment and consistently the re-oxygenation process significantly restored the expression level of Gal-1. Interestingly the stable knock-down of HIF-1a significantly down-regulated Gal-1 expression in HMCLs both in normoxic and hypoxic conditions. Thereafter, we explored the effect of persistent Gal-1 inhibition in MM cells and BM microenvironment cells on cell proliferation, survival and the transcriptional and pro-angiogenic profiles. An anti-Gal-1 Lentivirus shRNA was used for Gal-1 stable knock-down in HMCLs (JJN3-anti-Gal-1 and OPM-2-anti-Gal-1) and MSC cell lines (HS-5 and HMSC-Tert) and the Scramble lentiviral vector (JJN3-Scramble and OPM-2-Scramble) was used as the empty control vector. The stable inhibition of Gal-1 did not affect the proliferation rate and viability of both HMCLs and MSC cell lines. On the other hand Gal-1 inhibition by shRNA lentivirus significantly modified the transcriptional profiles of HMCLs and HS-5, evaluated by U133 Plus2.0 Arrays (Affymetrix®) either in normoxic or hypoxic or re-oxygenation conditions. Among the genes significantly modulated by Gal-1 inhibition in HMCLs, we found that pro-angiogenic (eg. CCL2, MMP9) and adhesion molecules (eg. MCAM and STEAP1) were down-regulated by Gal-1 suppression in both normoxic and hypoxic conditions as well as some putative anti-tumoral genes, including EGR1, SPARC and TGFBI, and anti-angiogenic ones, including SEMA3A, were up-regulated by Gal-1 inhibition. In line with these observations, we found that Gal-1 suppression by shRNA significantly decreased the pro-angiogenic proprieties of HMCLs by an in vitro angiogenesis assay. Finally, we found that mice, injected subcutaneously with JJN3-anti-Gal-1 and OPM-2-anti-Gal-1, showed a reduction in the weight and volume of the tumor burden compared to mice inoculated with the JJN3-Scramble and OPM-2-Scramble. Moreover, a significant reduction in the number of CD34 positive vessels X field was observed. In an intratibial mouse model, JJN3-anti-Gal-1, JJN3-Scramble and JJN3 wild type were injected: in the anti-Gal-1 group tumors grew in reduced number and size compared to the Scramble group, moreover JJN3 anti-Gal-1 mice developed fewer and smaller lytic lesions on x-ray compared to the controls. Overall our data indicate that Gal-1 is highly expressed by MM cells and those of the BM microenvironment and that its expression is regulated by hypoxia. Gal-1 shows a role in MM-induced angiogenesis and its inhibition in MM cells significantly reduced tumor growth in vivo, suggesting that Gal-1 is a potential new therapeutic target in MM. Disclosures Giuliani: Celgene Italy: Research Funding.

Description: Background- Multiple Myeloma (MM) is a hematologic malignancy strongly characterized by genomic instability, which promotes disease progression and drug resistance. We previously demonstrated that LIG3-dependent Alt-NHEJ repair is involved in genomic instability, drug resistance and survival of MM cells. On these premises, we investigated PARP1 as driver component of Alt-NHEJ pathway and new therapeutic target in MM. Materials and methods- Cell proliferation and apoptosis were evaluated with CellTiter-Glo assay and Annexin V staining. Alt-NHEJ repair was evaluated using EJ2-GFP. PARP1, Caspase 3, MYC and DNA Damage Response protein levels were analyzed by Western blot of whole protein extracts. In vivo anti-MM activity was evaluated in NOD-SCID mice bearing subcutaneous H929 and AMO-1 Bortezomib resistant (ABZB) xenografts, daily treated with Olaparib (Selleckchem) via oral gavage. Results - By interrogating public available datasets, we found significant correlation between higher mRNA expression of PARP1 and shorter survival of MM patients. On these findings, we investigated the effect of available PARP inhibitors (PARPi) on MM cell survival. We found that Olaparib, a clinically available PARPi induced a significant reduction of proliferation and clonogenic growth of MM cell lines at low micromolar concentrations. Importantly, Olaparib impaired viability of MM cell lines or primary malignant plasmacells co-cultured with stromal cells, thus overcoming the bone marrow microenvironment supportive effect for MM survival. As result of PARP-mediated Alt-NHEJ repair inhibition, anti-proliferative effects were associated to increase of DNA double-strand breaks (DSBs), activation of DNA damage response, cell cycle arrest and finally apoptosis. To identify predictive biomarkers for PARPi in MM, a published sensitivity gene expression signature was applied to our MM gene expression profiling (GEP) data. Interestingly, this signature was particularly enriched in TC2 MM and secondary plasma cell leukemia (PCL). Therefore, in order to evaluate concordantly modulated sets of genes that were possibly associated to PARPi signature in MM, PARPi-positive and PARPi-negative MM-TC2 cases were compared by GSEA analysis. Interestingly, groups of genes regulated by MYC or involved in DNA repair resulted among the most significantly up-regulated in PARPi-positive versus PARPi-negative MM-TC2 cases. Accordingly, MYC transcript reached the highest median expression levels in sPCL and HMCLs across PC dyscrasia groups, and in MM-TC2 class. Consistently U266 cells, which was quiet insensitive to PARP knockdown or PARP inhibitor Olaparib, were null for c-MYC as compared to multiple myeloma cell lines evaluated in this study. Conversely, as formal proof of our hypothesis, over-expression of c-MYC in U266 cells (MYC-OE) induced cell death upon PARP silencing or PARP inhibitor treatment. Notably, we found that c-MYC-PARP1 loop was also hyper-activated in Bortezomib resistant cells, thus confirming pivotal role of Alt-NHEJ repair in drug resistance development. Remarkably, to demonstrate the in vivo relevance of our findings, we showed that clinically available Parp-inhibitor Olaparib exerted a significant anti-MM activity on both Bortezomib sensitive (H929) and resistant (ABZB) MM cells injected in immunocompromised mice. Conclusion Taken together, our findings indicate that MM cells are dependent on PARP-mediated Alt-NHEJ repair pathway, which therefore represents a novel druggable target pathway in MYC-driven MM cells. Disclosures No relevant conflicts of interest to declare.

Description: Multiple myeloma (MM) is a fatal malignancy of clonal bone marrow plasma cells characterized by a high genomic instability increasing with disease progression. We describe here a genomic amplification at 17p11.2–p12, an unstable chromosomal region characterized by a large number of low copy repeats which have been proven to mediate deletion and duplication in several genomic disorders and amplifications in solid tumors. An approximately 5 Mb 17p11.2–p12 amplified region was detected in KMS-26 myeloma cell line by SNP microarray analysis. Further FISH mapping showed two unidentified amplified chromosomes as well as a complex pattern of rearranged chromosomes 17. The analysis of transcriptional profiles in a proprietary data base of myeloma cell lines, identified 12 significantly overexpressed genes in KMS–26 amplified region, including TNFRSF13B/TACI, COPS3, and NCOR1. The evaluation of their expression levels in a data base including 11 monoclonal gammopathy of uncertain significance, 121 MM and 9 plasma cell leukaemia, showed a significant overexpression of at least one gene in 13 patients. FISH analyses of these patients identified one MM carrying a 3.8 Mb amplified region and two MMs with gains specifically involving the TACI locus. Interestingly, the complete inactivation of TP53 at 17p13.1 was found in KMS–26 whereas a monoallelic loss was identifiable in two of the three patients carrying gain/amplification. Our data suggest that, as in the case of solid tumors, amplification/gain of the 17p11.2–p12 in MM could be mediated by the presence of repeats located in this region and may provide insights for defining novel candidate myeloma-associated genes.

Description: Proteasome inhibitors (PI) are extensively used for the therapy of multiple myeloma (MM) and mantle cell lymphoma. However, patients continuously relapse or are intrinsically resistant to this class of drugs. Here, to identify targets that synergize with PI, we carried out a functional screening in MM cell lines using a short hairpin RNA library against cancer driver genes. Isocitrate dehydrogenase 2 (IDH2) was identified as a top candidate, showing a synthetic lethal activity with the PI carfilzomib (CFZ). Combinations of US Food and Drug Administration–approved PI with a pharmacological IDH2 inhibitor (AGI-6780) triggered synergistic cytotoxicity in MM, mantle cell lymphoma, and Burkitt lymphoma cell lines. CFZ/AGI-6780 treatment increased death of primary CD138+ cells from MM patients and exhibited a favorable cytotoxicity profile toward peripheral blood mononuclear cells and bone marrow–derived stromal cells. Mechanistically, the CFZ/AGI-6780 combination significantly decreased tricarboxylic acid cycle activity and adenosine triphosphate levels as a consequence of enhanced IDH2 enzymatic inhibition. Specifically, CFZ treatment reduced the expression of nicotinamide phosphoribosyltransferase (NAMPT), thus limiting IDH2 activation through the NAD+-dependent deacetylase SIRT3. Consistently, combination of CFZ with either NAMPT or SIRT3 inhibitors impaired IDH2 activity and increased MM cell death. Finally, inducible IDH2 knockdown enhanced the therapeutic efficacy of CFZ in a subcutaneous xenograft model of MM, resulting in inhibition of tumor progression and extended survival. Taken together, these findings indicate that NAMPT/SIRT3/IDH2 pathway inhibition enhances the therapeutic efficacy of PI, thus providing compelling evidence for treatments with lower and less toxic doses and broadening the application of PI to other malignancies.

Description: Multiple myeloma (MM) is characterized by a high genomic instability that involves both ploidy and structural rearrangements. Nearly half of MM tumors are non-hyperdiploid and are frequently associated with 13q deletion and chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus on chromosome 14q32. The remaining tumors are hyperdiploid, showing low prevalence of both IGH translocations and chromosome 13 deletions. Our study was aimed at providing insights into the genomic heterogeneity associated with plasma cell neoplasms by defining the genome-wide pattern of genetic lesions in a representative and stratified panel of MM patients. To this end, genome-wide profiling data of 45 plasma cell dyscrasia patients (41 MM and 4 plasma cell leukemia) were generated on GeneChip® Human Mapping 50K Xba SNP arrays, and the local DNA copy number variations were calculated using the DNAcopy Bioconductor package. The patients were clustered using the non-negative matrix factorization (NMF) algorithm to identify, within the natural grouping of profiles, the strongest clusters on the basis of their genomic characteristics. We identified three consistent clusters, characterized byrecurrent gains of odd-chromosomes, suggestive of the hyperdiploid status (Group A),high frequency of chromosome 13 deletion and 1q gains (Group B), orhigh frequency of chromosomes 13, 14, 16 and 22 deletions and losses of 1p and 4p regions, together with some cases showing 1q gains (Group C). To determine whether peculiar transcription fingerprints characterized these groups, gene expression profiles of 40 out of 45 corresponding samples generated on GeneChip® HG-U133A arrays were analyzed using the Prediction Analysis of Microarray (PAM) software. The multi-class analysis identified 229 transcripts (corresponding to 195 genes), which specifically marked the three groups. In particular, Group A was characterized by the overexpression of genes involved in the translational machinery or thought to be involved in MM pathogenesis such as the HGF, the tumor necrosis factor ligand TRAIL, DKK1, and c-KIT. Upregulation of the CKS1B gene was present in Group B and C, most likely reflecting the high frequencies of 1q gains in tumors within group B and C and its consequent deregulation. Group C was marked by the specific downregulation of genes mainly mapped to 1p arm: AMPD1, CSDE1, AKR1A1 and the PRKACB kinase, suggesting a relationship with the recurrent 1p loss within the group. Our data further supported the notion that structural abnormalities in multiple myeloma are associated with gene expression imbalances, and provide novel analytical approaches for the identification of genetic lesions and molecular patterns of the disease.

Description: The malignant plasma cells (PC) dependence on the bone marrow (BM) microenvironment is a main feature of multiple myeloma (MM), mainly due to high expression of cell adhesion molecules including CD44, CD54 (ICAM1), CD56, CD49d and chemokines receptors such as CXCR4. At the end stage of the disease, a rare MM complication, that may occurs, is a secondary extramedullary involvement without a leukemic phase and, among the extramedullary localizations, the skin is one of the possible sites, due to the physiological homing of PCs. However, the mechanisms behind the extramedullary spread are not completely understood. In a patient with refractory resistant MM, who has developed a cutaneous localization after 16 months from the diagnosis under Bortezomib (BOR) treatment, we analyzed the gene expression profiles (GEPs) by GeneChip U133 Plus 2.0 (Affymetrix), the immunophenotypic and immunohistochemistry (IHC) profiles of MM cells, across the course of the disease. To confirm the results, IHC profiles on selected molecules were then analyzed in other six MM patients with secondary skin involvement without PC leukemia. On BM CD138+ PCs at diagnosis and BM relapse, by GEP analysis, 742 genes resulted differentially expressed. Moreover, a more stringent analysis identified 19 down-regulated and 42 up-regulated genes; data were confirmed by Real Time PCR on selected genes mainly involved in PC homing. At the BM relapse, we found that MM cells expressed CXCR4 and were negative for CD56; moreover, a significant down-regulation of ICAM1 (CD54) and ALCAM was observed together with the up-regulation of MAGE family genes, DKK1 and SEMA3A in the BM relapse sample compared to diagnosis one. At the cutaneous involvement development, 4 months after BM relapse, by IHC analysis, the MM cells localized in the skin showed the expression of both CD56 and CD54. On the other hand, the BM MM cells immunophenotype confirmed the presence of CD56 but showed the lack of CXCR4 expression. IHC for CD54, CD56 and CXCR4 expression was then performed on both skin and bone biopsies of the other patients analyzed. To go further inside on the role of CXCR4 we treated with BOR (5-10nM) for 24-48h human myeloma cell lines (HMCLs) RPMI-8226 and U266, known to be resistant to BOR, evaluating the CXCR4 expression at both mRNA and protein level, by real-time PCR and by cytofluorimetry, respectively. CXCR4 resulted downregulated after BOR treatment in BOR resistant HMCLs. Consistently, the loss of CXCR4 expression was recently associated to BOR resistance and extramedullary disease in a mouse model of MM suggesting that CXCR4 loss can be correlated with BOR resistance and PCs mobilization from BM, leading to an extramedullary disease. In conclusion, our data indicate that the loss of CXCR4 expression by MM cells across the disease progression, together with CD54 and CD56 down-regulation and their re-acquisition at the extramedullary site, are involved driving the escape of PCs from BM to the extramedullary localization in the skin in the context of Bortezomib resistance. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4040 Multiple myeloma (MM) is a clonal proliferation of malignant plasma cells (PCs) characterized by a marked genomic instability. Primary plasma-cell leukemia (pPCL) is an aggressive, rare variant of plasma cell dyscrasia characterized by extra-medullary proliferation of PCs, high genomic instability and very poor prognosis. To date, global genomics studies in pPCL are still limited. Highly purified PCs were obtained from 17 previously untreated pPCL patients, recruited in an open-label, exploratory, single-arm, two-stage study from the GIMEMA myeloma network aimed at the evaluation of safety and antitumor activity of the immunomodulatory agent lenalidomide in combination with low dose dexamethasone in previously untreated pPCL. All the samples were characterized for the main chromosomal aberrations by Fluorescence in-situ hybridization (FISH). Specifically, 13q and 17p deletions have been identified in 12/17 (70.6%) and 8/17 (47.1%) cases, respectively; the presence of t(11;14) was found in 4/17 patients (23.5%), t(4;14) in only 1/17 cases (5.9%) and MAF translocations in 8/17 (47.1%). To further investigate the genomic complexity of pPCL, we analyzed a subset of 13 samples by means of an integrative approach using different high-throughput microarray technologies: Human Mapping 250K Nsp SNP-array (Affymetrix) for the detection of copy number alterations (CNA), Gene 1.0 ST array (Affymetrix) for transcriptional profiling and miRNA Microarray v2 (Agilent) for the global miRNA expression. SNP-array data were concordant with FISH results for the detection of the main chromosomal aberrations, i.e. 13q (10/13=77%) and 17p (7/13=58.3%) deletions. The most recurrent CNA specifically identified by SNP-array was represented by 1q gain (8/13=61.5%); in addition. losses involving chromosomes 1p (5/13=38.5%), 8p (4/13=30.8%), 14q (5/13=38.5%), 16q (5/13=38.5%), gains affected 7q (4/13=30.8%) and 19p (4/13=30.8%) and one amplification at 17q21 in 6/13 pPCL (46.2%) were detected. One case displayed a near tetraploid karyotype and, interestingly, another one showed a hyperdiploid pattern. Mapping information was integrated with the gene expression and miRNA profiles of the tumor samples. A non-parametric analysis (Kendall's tau correlation at a p-value

Description: Abstract 448 The increase of osteoclast formation and activation occurring into the bone marrow (BM) area of myeloma cells infiltration is the hallmark of multiple myeloma (MM). The increase of the RANKL/OPG ratio in BM stromal cells (BMSCs) and osteoprogenitor cells, induced by MM cells through the cell-to-cell contact, is critically involved in MM-induced osteoclast formation. In addition, MM cells also up-regulate RANKL expression and secretion by activated T in the MM BM microenvironment. Soluble factors such as CCL3/MIP-1α, IL-3 and IL-7 produced by MM cells contribute to the increase of osteoclast formation either directly or indirectly through RANKL stimulation. Recent data have suggested that thalidomide and the immunomodulatory drugs (IMiDs®) lenalidomide directly inhibit osteoclast formation and maturation. In the present study we have investigated the potential effects of lenalidomide and the more potent IMiD® pomalidomide on MM-induced osteoclast formation. First in a cell-to-cell contact co-culture system we found that both IMiDs® at concentration ranging from 2 to 100 μM significantly blunted RANKL secretion by human BMSC/osteoprogenitor cells induced by MM cells decreasing the RANKL/OPG ratio level with a more potent effect of pomalidomide as compared to lenalidomide. Consistently the pro-osteoclastogenic property of the conditioned medium of MM cells co-cultured with BMSC/osteoprogenitor cells was reduced in the presence of IMiDs®. On the other hand, we did not find any significant inhibitory effect of both drugs neither on the production of soluble pro-osteoclastogenic factors by MM cells as CCL3/MIP-1α, IL-3 and IL-7 nor on RANKL expression and secretion by T lymphocytes. To go further inside to the capacity of IMiDs® to blunt MM-induced RANKL/OPG ratio, we performed a microarray analysis (using Affymetrix, GeneChip®, HG-U133Plus2.0 platform) to investigate the effect of lenelidomide and pomalidomide on the transcriptional profile of both MM cells and BMSCs. We found that 40 and 83 genes were significantly modulated by lenalidomide and pomalidomide, respectively, in MM cells Interestingly, among the genes significantly modulated by IMiDs® we identified the downregulation of the adhesion molecules ITGA4 (CD49d), ITGA8 and ICAM2 (CD102). In human BMSCs, 71 genes and 214 genes were significantly modulated by lenalidomide and pomalidomide, respectively, including those belonging to focal adhesion, cell cycle, BMP2, TGF-beta and IL-6 signaling pathways. Finally, using flow cytometry we confirmed the capacity of lenalidomide and pomalidomide to inhibit the expression of adhesion molecules as CD49d by MM cells co-cultured either in presence or absence of BMSCs showing that this effect is critically involved in the inhibition of RANKL/OPG ratio by IMiDs®. In conclusion our data strongly suggest that lenalidomide and pomalidomide inhibits MM-induced osteoclast formation through the inhibition of RANKL/OPG ratio targeting the expression of adhesion molecules by MM cells. Disclosures: Giuliani: Celgene: Research Funding.