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Accumulating evidence sustains glial cells as critical players during central nervous system (CNS) development, homeostasis and disease. Olfactory ensheathing cells (OECs), a type of specialized glia cells sharing properties with both Schwann cells and astrocytes, are of critical importance in physiological condition during olfactory system development, supporting its regenerative potential throughout the adult life. These characteristics prompted research in the field of cell-based therapy to test OEC grafts in damaged CNS. Neuroprotective mechanisms exerted by OEC grafts are not limited to axonal regeneration and cell differentiation. Indeed, OEC immunomodulatory properties and their phagocytic potential encourage OEC-based approaches for tissue regeneration in case of CNS injury. Herein we reviewed recent advances on the immune role of OECs, their ability to modulate CNS microenvironment via bystander effects and the potential of OECs as a cell-based strategy for tissue regeneration.

Glial activation and related neuroinflammatory processes play a key role in the aging and progression of Alzheimer's disease (AD). CHI3L1/ YKL40 is a widely investigated chitinase in neurodegenerative diseases and recent studies have shown its involvement in aging and AD. Nevertheless, the biological function of CHI3L1 in AD is still unknown. Here, we collected microarray datasets from the National Center for Biotechnology Information (NCBI) brain samples of not demented healthy controls (NDHC) who died from causes not attributable to neurodegenerative disorders (n = 460), and of deceased patients suffering from Alzheimer's disease (AD) (n = 697). The NDHC and AD patients were stratified according to CHI3L1 expression levels as a cut-off. We identified two groups both males and females, subsequently used for our statistical comparisons: the high CHI3L1 expression group (HCEG) and the low CHI3L1 expression group (LCEG). Comparing HCEG to LCEG, we attained four signatures according to the sex of patients, in order to identify the healthy and AD brain cellular architecture, performing a genomic deconvolution analysis. We used neurological signatures (NS) belonging to six neurological cells populations and nine signatures that included the main physiological neurological processes. We discovered that, in the brains of NDHC the high expression levels of CHI3L1 were associated with astrocyte activation profile, while in AD males and females we showed an inflammatory profile microglia-mediated. The low CHI3L1 brain expression levels in NDHC and AD patients highlighted a neuronal activation profile. Furthermore, using drugs opposing CHI3L1 transcriptomic signatures, we found a specific drug profile for AD males and females characterized by high levels of CHI3L1 composed of fostamatinib, rucaparib, cephaeline, prednisolone, and dinoprostone. Brain levels of CHI3L1 in AD patients represent a biological signature that allows distinguishing between males and females and their likely cellular brain architecture.

Lactic acidosis has been reported in solid tumor microenvironment (TME) including glioblastoma (GBM). In TME, several signaling molecules, growth factors and metabolites have been identified to induce resistance to chemotherapy and to sustain immune escape. In the early phases of the disease, microglia infiltrates TME, contributing to tumorigenesis rather than counteracting its growth. Insulin-like Growth Factor Binding Protein 6 (IGFBP6) is expressed during tumor development, and it is involved in migration, immune-escape and inflammation, thus providing an attractive target for GBM therapy. Here, we aimed at investigating the crosstalk between lactate metabolism and IGFBP6 in TME and GBM progression. Our results show that microglia exposed to lactate or IGFBP6 significantly increased the Monocarboxylate transporter 1 (MCT1) expression together with genes involved in mitochondrial metabolism. We, also, observed an increase in the M2 markers and a reduction of inducible nitric oxide synthase (iNOS) levels, suggesting a role of lactate/IGFBP6 metabolism in immune-escape activation. GBM cells exposed to lactate also showed increased levels of IGFBP6 and vice-versa. Such a phenomenon was coupled with a IGFBP6-mediated sonic hedgehog (SHH) ignaling increase. We, finally, tested our hypothesis in a GBM zebrafish animal model, where we observed an increase in microglia cells and igfbp6 gene expression after lactate exposure. Our results were confirmed by the analysis of human transcriptomes datasets and immunohistochemical assay from human GBM biopsies, suggesting the existence of a lactate/IGFBP6 crosstalk in microglial cells, so that IGFBP6 expression is regulated by lactate production in GBM cells and in turn modulates microglia polarization.

Inflammation represents a key feature and hallmark of tumor microenvironment playing a major role in the interaction with mesenchymal stromal cells (MSC) in cancer progression. The aim of the present study was to investigate the crosstalk between MSCs and myeloma cells (MM) in the pro-inflammatory microenvironment promoting immune evasion and tumor growth. MSC were collected from patients with diagnosis of MGUS (n = 10), smoldering myeloma (n = 7), multiple myeloma at diagnosis (n = 16), relapse (n = 5) or refractory (n = 3), and from age-matched healthy controls (HC, n = 10) and cultured with peripheral blood mononucleated cells (PBMC) from healthy volunteer donors. Similarly to MM, we showed that MSC from smoldering multiple myeloma (SMM) patients activated neutrophils and conferred an immunosuppressive and pro-angiogenic phenotype. Furthermore, co-cultures of plasma cells (PC) and HC-MSC suggested that such activation is driven by MM cells through the switching into a pro-inflammatory phenotype mediated by toll-like receptor 4 (TLR4). These results were further confirmed using a zebrafish as an immunocompetent in vivo model, showing the role of MM–MSC in supporting PCs engraftment and Th2 response. Such effect was abolished following inhibition of TLR4 signaling in MM–MSC before co-injection with PC. Moreover, the addition of a TLR4 inhibitor in the co-culture of HC-MSC with MM cells prevented the activation of the pro-tumor activity in PC-educated MSC. In conclusion, our study provides evidence that TLR4 signaling plays a key role in MSC transformation by inducing a pro-tumor phenotype associated with a permissive microenvironment allowing immune escape and tumor growth.

Although the epidemic caused by SARS-CoV-2 callings for international attention to develop new effective therapeutics, no specific protocol is yet available, leaving patients to rely on general and supportive therapies. A range of respiratory diseases, including pulmonary fibrosis, have been associated with higher iron levels that may promote the course of viral infection. Recent studies have demonstrated that some natural components could act as the first barrier against viral injury by affecting iron metabolism. Moreover, a few recent studies have proposed the combination of protease inhibitors for therapeutic use against SARS-CoV-2 infection, highlighting the role of viral protease in virus infectivity. In this regard, this review focuses on the analysis, through literature and docking studies, of a number of natural products able to counteract SARS-CoV-2 infection, acting both as iron chelators and protease inhibitors.

Motoneuronal loss is the main feature of amyotrophic lateral sclerosis, although pathogenesis is extremely complex involving both neural and muscle cells. In order to translationally engage the sonic hedgehog pathway, which is a promising target for neural regeneration, recent studies have reported on the neuroprotective effects of clobetasol, an FDA-approved glucocorticoid, able to activate this pathway via smoothened. Herein we sought to examine functional, cellular, and metabolic effects of clobetasol in a neurotoxic mouse model of spinal motoneuronal loss. We found that clobetasol reduces muscle denervation and motor impairments in part by restoring sonic hedgehog signaling and supporting spinal plasticity. These effects were coupled with reduced pro-inflammatory microglia and reactive astrogliosis, reduced muscle atrophy, and support of mitochondrial integrity and metabolism. Our results suggest that clobetasol stimulates a series of compensatory processes and therefore represents a translational approach for intractable denervating and neurodegenerative disorders.

To understand neutrophil impairment in the progression from MGUS through active MM, we investigated the function of mature, high-density neutrophils (HDNs), isolated from peripheral blood. In 7 MM, 3 MGUS and 3 healthy subjects by gene expression profile, we identified a total of 551 upregulated and 343 downregulated genes in MM-HDN, involved in chemokine signaling pathway and FC-gamma receptor mediated phagocytosis conveying in the activation of STAT proteins. In a series of 60 newly diagnosed MM and 30 MGUS patients, by flow-cytometry we found that HDN from MM, and to a lesser extend MGUS, had an up-regulation of the inducible FcγRI (also known as CD64) and a down-regulation of the constitutive FcγRIIIa (also known as CD16) together with a reduced phagocytic activity and oxidative burst, associated to increased immune-suppression that could be reverted by arginase inhibitors in co-culture with lymphocytes. In 43 consecutive newly-diagnosed MM patients, who received first-line treatment based on bortezomib, thalidomide and dexamethasone, high CD64 could identify at diagnosis patients with inferior median overall survival (39.5 versus 86.7 months, p = 0.04). Thus, HDNs are significantly different among healthy, MGUS and MM subjects. In both MGUS and MM neutrophils may play a role in supporting both the increased susceptibility to infection and the immunological dysfunction that leads to tumor progression.

Background In Hodgkin Lymphoma (HL), neoplastic cells orchestrate an inflammatory microenvironment leading to sterile inflammation, T-cell anergy, and immune deficiency. Our group showed that in HL patients the aminoacid degrading enzyme Arginase-1 is increased, associated with poor outcome, and leads to arginine (Arg) deprivation. However, how the reduction of Arg in the extracellular milieu of the tumor microenvironment can contribute to neoplastic cell fitness is largely unknown. Aims To detect the adaptive response (via evaluation of global transcriptome and metabolome changes) in human HL cell lines exposed to Arg deprivation. Methods To better understand the impact of extra-cellular Arg1 deprivation on the metabolome of human cHL cells, four human cHL cell lines (L428, L540, HDMYZ and KM-H2) were individually cultured with customized complete media or lacking or Arg (R0), supplemented with 10% dialyzed fetal bovine serum, in six independent experiments. After 48 hours of culture, the cells were collected for global metabolomic analysis, by gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) platforms by Metabolon Inc and transcriptome profiling by Illumina platform. Following normalization to DNA concentration, log transformation, and imputation of missing values, if any, with the minimum observed value for each compound, Welch's two-sample t-test was used to identify biochemicals that differed significantly between experimental groups. Results While Arg deprivation did not affect cell viability but delayed cell cycle due to arrest in G2 phase in all tested cell lines, the effect of Arg deficiency on the cellular metabolome depended largely on the cell type examined with L428 and KMH2 cells having significantly changed metabolomes. Pyruvate was significantly higher in the KMH2 cells deprived of Arg compared to controls. Conversely, lactate was significantly lower, with increased levels of long-chain saturated fatty acids and long-chain polyunsaturated fatty acids (PUFAs). Taken together the metabolomics changes suggested that specific-amino acid deficiency can lead to an increase in free fatty acids synthases to preserve cytoplasmatic and mitochondrial membrane dynamics. Consistent with a metabolic rewiring to maintain mitochondrial integrity (the pyruvate is an important intermediary in the conversion of carbohydrates into fatty acids), the adaptive response was associated to increased oxidative stress, as suggested by of reduced glutathione in KMH2 cells, depletion of gamma-glutamylcysteine, increased cystine, the oxidative product of cysteine, and methionine sulfoxide (an oxidation product of methionine). Gene set enrichment analysis (GSEA) showed deep transcriptome rearrangements in KMH2 and HDMYZ cell lines, involving upregulation of genes required for the unfolded protein response (UPR, including XBP1, EIF2S1, EIF4A2, EIF4A3, ATF3, ATF4, DDIT4, EDEM1, GADD45B, SQSTM1, HMOX), NF-kB response to TNF (including RAF1, TNF, LIF, NKBIA, SGK1, BIRC3, ICAM1, BCL6, IL6, RELA, CDKN1A), p53 pathway and networks (including CDKN2B, STOM, TRAF4, RRAD, SESN1, FOXO3, SERPINB5, JAG2) and proteosome degradation (HSPA4, PSMD11, PSMD13, PSMD2, PSMA5, PSMA7, PSMC4), with a minimal effect on metabolism features, except the upregulation of genes involved in lactate generation and degradation. All lines tested showed down-regulation of CCNI2, LCROL, MKI67, NCAPG, PEX10 and UFSP2, suggesting that early response to arginine deprivation includes modulation of UFMylation pathway, the most recently discovered post-translational protein modification system, whose biological function is largely unknown. Conclusions The removal of Arg from L428 and KMH2 resulted in changes in the specific-amino acid-related metabolites. The adaptive response to Arg-depleted environment increases oxidative stress and promotes a shift in glucose use in the attempt to preserve mitochondrial function. The cell-cycle arrest in G2, the increase of pyruvate availability and the upregulation of proteasome function via upregulation of the UFMylation pathway suggest the dependency of HL cell lines on mitochondrial function integrity. Quantity and function of mitochondria network can play a major role in selecting the fittest clones, a metabolic pathway that should be explored as novel non -synthetic lethal targets. Disclosures Martinelli: Stemline Therapeutics: Consultancy; Roche: Consultancy; Astellas: Consultancy, Speakers Bureau; Daichii Sankyo: Consultancy; Pfizer: Consultancy, Speakers Bureau; Incyte: Consultancy; Abbvie: Consultancy; Celgene /BMS: Consultancy, Speakers Bureau; Jazz Pharmaceuticals: Consultancy. Di Raimondo: Pfizer: Honoraria; Jazz Pharmaceutical: Honoraria; Amgen: Honoraria; AbbVie: Honoraria; Bristol Myers Squibb: Honoraria; Janssen Pharmaceuticals: Honoraria.

Mesenchymal stromal cells (MSCs) within the protective microenvironment of multiple myeloma (MM) promote tumor growth, confer chemoresistance and support metabolic needs of plasma cells (PCs) also transferring mitochondria. In this scenario, heterocellular communication and dysregulation of critical signaling axes are among the major contributors to progression and treatment failure. As metabolic rewiring is involved in the regulation of MSC phenotype, we first analyzed metabolic profile of healthy control (HC-) and MM-MSCs. NAD +/NADH ratio was decreased in MM-MSCs (n=8) as compared with HC-MSCs (n=4, p In the cell-to-cell contact the gap junction-forming protein CX43 has been found critical for mitochondria uptake in lung and brain injury and it also can regulate CXCL12 secretion by MSCs. We found that MM-MSCs showed a significantly up-regulated CXCL12 expression as compared to HC-MSCs (p In conclusion, we have shown that MM-MSCs are relatively low dependent on mitochondria metabolism and are inclined to transfer mitochondria to MM tumor cells. Furthermore, tumor PCs increase the expression of CX43 in MSCs leading to an increased levels of CXCL12 and stimulation of its corresponding receptor expressed on MM cells. The resulting CX43/CXCL12/CXCR4 interplay enhances mitochondrial trafficking from MSCs to myeloma PCs and can protect cancer cells against anti-myeloma agents. Understanding pro-tumorigenic phenotype of MSCs and mechanisms of adhesion and heterocellular communication favoring their interaction with cancer PCs, will allow to manipulate critical pathways, including CXCL12/CXCR4 axis, thus improving disease outcome. Disclosures Di Raimondo: Pfizer: Honoraria; AbbVie: Honoraria; Bristol Myers Squibb: Honoraria; Jazz Pharmaceutical: Honoraria; Janssen Pharmaceuticals: Honoraria; Amgen: Honoraria.

// Lucia Longhitano 1 , Carlo Castruccio Castracani 1 , Daniele Tibullo 1 , Roberto Avola 1 , Maria Viola 1 , Giuliano Russo 1 , Orazio Prezzavento 4 , Agostino Marrazzo 4 , Emanuele Amata 4 , Michele Reibaldi 2 , Antonio Longo 2 , Andrea Russo 2 , Nunziatina Laura Parrinello 3 and Giovanni Li Volti 1, 5 1 Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy 2 Department of Ophthalmology, University of Catania, Catania, Italy 3 Regional Reference Center for Rare Diseases, Clinical Division of Hematology and Transplantation, PO Ferrarotto Hospital, Azienda Ospedaliera-Universitaria Policlinico-Vittorio Emanuele, Via Citelli, Catania, Italy 4 Department of Drug Sciences, University of Catania, Catania, Italy 5 Euromediterranean Institute of Science and Technology, Palermo, Italy Correspondence to: Giovanni Li Volti, email: livolti@unict.it Keywords: uveal melanoma, sigma receptors, apoptosis, autophagy, (+)-pentazocine Received: June 16, 2017 Accepted: July 13, 2017 Published: July 25, 2017 ABSTRACT Uveal melanoma is the most common primary intraocular tumor in adults, with about 1200–1500 new cases occurring per year in the United States. Metastasis is a frequent occurrence in uveal melanoma, and outcomes are poor once distant spread occurs and no clinically significant chemotherapeutic protocol is so far available. The aim of the present study was to test the effect of various σ 1 and σ 2 receptor ligands as a possible pharmacological strategy for this rare tumor. Human uveal melanoma cells (92.1) were treated with various concentrations of different σ 2 ligands (haloperidol and haloperidol metabolite II) and σ 1 ligand ((+)-pentazocine) at various concentrations (1, 10 and 25 μM) and time points (0, 4 h, 8 h, 24 h and 48 h). Cell proliferation and migration were evaluated respectively by continuous cell monitoring by xCELLigence analysis, clonogenic assay and wound healing. Apoptosis and autophagy were also measured by cytofluorimetric and microscopy analysis. Our results showed that σ 2 receptor ligands significantly reduced cell proliferation whereas (+)-pentazocine exhibited opposite results. All tested ligands showed significant decrease in cell migration. Interestingly, both σ 1 and σ 2 receptor ligands showed significant increase of autophagy and apoptosis at all concentrations. Taken all together these results suggest that sigma receptors mediates opposite biological effects but they also share common pharmacological effect on apoptosis and autophagy in uveal melanoma. In conclusion, these data provide the first evidence that sigma receptors may represent a “druggable” target to develop new chemotherapic agent for uveal melanoma.

Heme oxygenase (HO)-1 catalyzes the conversion of heme to biliverdin, iron an d carbon monoxide. In myeloma plasma cells, its expression has been demonstrated to increase during bortezomib (BTZ) treatment and localize into the nucleus conferring drug resistance. Recently, our group demonstrated that BTZ also induces up-regulation of Toll like receptor 4 (TLR4) which acts as a stress-responsive mechanism protecting mitochondria during BTZ exposure and sustaining mitochondrial metabolism. Since two studies independently demonstrated that both HO-1 and TLR4 protect myeloma cells from BTZ-induced apoptotic signals, specific functional connections between these two proteins were considered herein. MM cell lines were treated with BTZ alone or in combination with TAK-242, a selective inhibitor of TLR4. We observed a significant increase of apoptosis in TAK-242/BTZ treated cells compared to BTZ alone. Drug combination also led to higher mitochondrial depolarization and decreased mitochondrial mass evaluated using flow cytometry. Accordingly, TAK-242/BTZ treatment act ivated mitophagy as demonstrated by evaluating co-localization of the autophagosome marker LC3 with mitochondria using confocal microscopy. Since it is known BTZ treated cells increased HO-1 expression as protective mechanism, we next evaluated if BTZ combination with TAK-242 could affect HO-1 expression. Western blot showed a down-regulation of HO-1 after TAK-242/BTZ treatment. Immunofluorescence analysis confirmed that drug combination decreased nuclear HO-1 and increased its cytoplasmic localization compared to BTZ alone. To address this controversy, we administered tin protoporphyrin (SnPP), a well-characterized HO-1 enzymatic inhibitor, alone or in combination with BTZ. Interestingly, SnPP/BTZ treated cells showed lower expression of TLR4 compared to BTZ treated ones. To better investigate if HO-1 enzymatic activity could regulate TLR4 expression, MM cells were exposed to hemin, an inducer of HO-1. We observed a significant up-regulation of TLR4 and NF-kB nuclear localization. Treating cells with rapid or slowly carbon monoxide-releasing molecules (CORM-3 and CORM-A1), an increase of TLR4 expression was observed after 3h with the consequent activation of p-p38, p-ERK and NF-kB nuclear translocation. Moreover, silencing HO-1 confirmed its role in the regulation of TLR4 expression. Moreover, compared to U266 cells, shHO-1/U266 cells howed higher apoptosis after treatment with BTZ, confirming that HO-1/TLR4 signaling protect MM cells from BTZ-induced apoptosis. Our data demonstrate that a functional regulatory link exists between HO1 and TLR4 which in turn impact on drug response. Specifically, inhibition of HO-1/TLR4 axis augmented cytotoxicity of BTZ against MM cells. In conclusion the HO-1/TLR4 axis is involved in BTZ mediated chemoresistance thus providing an important tool to improve the clinical outcome of MM patients resistant to BTZ. Disclosures Romano: Takeda: Honoraria; Novartis: Honoraria. Puglisi:Amgen: Honoraria. Conticello:Amgen, Takeda, Janssen: Honoraria. Palumbo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Di Raimondo:Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen, Takeda, Novartis: Honoraria; GILEAD, Incyte: Research Funding.

Background Tryptophan (trp) is an essential aminoacid, required for de novo NAD+ synthesis. Our previous work showed that the microenvironment in classical Hodgkin Lymphoma is characterized by dysfunctional neutrophils and myeloid derived suppressor cells that produce a trp-degrading enzyme indoelamine deoxygenase (IDO-1), lowering the amount of trp. Aims To detect metabolomics changes in human cHL cell lines exposed in vitro to tryptophan deprivation, an amino acid involved in immune dysregulation and generation of anergic and tolerogenic T- cells. Methods In order to better understand the impact of extra-cellular IDO1 increase on the metabolome of human cHL cells, three human cHL cell lines (L428, L540 and KM-H2) were individually cultured with customized complete media or media lacking tryptophan (W0), +10% dyalised fetal bovum serum, in six independent experiments. After 48 hours of culture the cells were collected for global metabolomic analysis, by gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) platforms by Metabolon Inc. Following normalization to DNA concentration, log transformation and imputation of missing values, if any, with the minimum observed value for each compound, Welch's two-sample t-test was used to identify biochemicals that differed significantly between experimental groups (Table 1). Results The lack of tryptophan in media had a profound effect on the cell metabolome in 2 cell lines, KMH2 and L428 cells, while L540 cell line was pretty resilient (Table 1). In all cell lines, the removal of tryptophan from the media resulted in significantly lower levels of tryptophan. Kynurenine, the metabolic product of IDO-1 action on tryptophan, was lower in all cells, but did not reach significance in the L540 cells, whereas it trended lower in L428 and was significantly lower in the KMH2 compared to controls. Indolelactate, another major tryptophan metabolite was also significantly lower in the L428 and KMH2 cells lines compared to controls. Glucose uptake and aerobic glycolysis are frequently upregulated in tumor cells to support energy needs and provide biosynthetic precursors (e.g. pentose phosphate pathway intermediates for nucleotide synthesis). Known as the Warburg effect, this process of reliance on glucose for energy results in high levels of lactate production. We found that trp deficiency lead to lower levels of the hexose diphosphates (fructose 1,6-diphosphate/glucose 1,6-diphosphate/myo-inositol diphosphates isobar) and dihydroxyacetone phosphate, suggesting the revert of Warburg effect due to reduced bio-energetic requirements for proliferation. In line with this observation, culture in trp deficient media resulted in increased levels of long chain saturated fatty acids and long chain polyunsaturated fatty acids (PUFAs), suggesting that specific-amino acid deficiency leads to an increase in uptake of free fatty acids from the media, to preserve membrane dynamics. Since prolonged trp deprivation (up to 10 days) delayed cell cycle length without affecting proliferation or changes in intracellular amount of NAD+, we investigated changes in mitochondrial membranes network to explain these findings. Trp deprivation induced the rearrangement of the mitochondrial network at 48 hours, with more fission than fusion, as suggested by increased expression of Fis1 and Drp1 and reduced expression of Tfam and Opa1, without affecting significantly mitochondrial mass and depolarization. This adaptive response was associated to increased oxidative stress, as suggested by of reduced glutathione (GSH) and oxidized glutathione (GSSG) in the L540 and KMH2 cells, depletion of gamma-glutamylcysteine, increased cystine, the oxidative product of cysteine, and methionine sulfoxide (an oxidation product of methionine). Conclusions The removal of trp from L428 and KMH2 resulted in changes in the specific-amino acid related metabolites. The adaptive response to trp-depleted microenvironment can revert the Warburg effect, promoting a shift in the glucose use in the futile attempt to preserve mitochondrial function, and increase oxidative stress. Quantity and function of mitochondria network can play a major role in selecting the fittest clones, a metabolic pathway that should be explored as novel non -synthetic lethal targets. Disclosures Puglisi: Amgen:Honoraria.Di Raimondo:Takeda:Consultancy, Honoraria;GILEAD, Incyte:Research Funding;Amgen, Takeda, Novartis:Honoraria;Celgene:Consultancy, Honoraria;Janssen:Consultancy, Honoraria;GSK:Consultancy, Honoraria;Amgen:Consultancy, Honoraria.Romano:Takeda:Honoraria;Novartis:Honoraria.

Inflammation represents a key feature and hallmark of tumor microenvironment playing a major role in the interaction with mesenchymal stromal cells (MSC) in cancer progression. The aim of the present study was to investigate the crosstalk between MSCs and myeloma cells (MM) in the pro-inflammatory microenvironment promoting immune evasion and tumor growth. MSC were collected from patients with diagnosis of MGUS (n = 10), smoldering myeloma (n = 7), multiple myeloma at diagnosis (n = 16), relapse (n = 5) or refractory (n = 3), and from age-matched healthy controls (HC, n = 10) and cultured with peripheral blood mononucleated cells (PBMC) from healthy volunteer donors. Similarly to MM, we showed that MSC from smoldering multiple myeloma (SMM) patients activated neutrophils and conferred an immunosuppressive and pro-angiogenic phenotype. Furthermore, co-cultures of plasma cells (PC) and HC-MSC suggested that such activation is driven by MM cells through the switching into a pro-inflammatory phenotype mediated by toll-like receptor 4 (TLR4). These results were further confirmed using a zebrafish as an immunocompetent in vivo model, showing the role of MM-MSC in supporting PCs engraftment and Th2 response. Such effect was abolished following inhibition of TLR4 signaling in MM-MSC before co-injection with PC. Moreover, the addition of a TLR4 inhibitor in the co-culture of HC-MSC with MM cells prevented the activation of the pro-tumor activity in PC-educated MSC. In conclusion, our study provides evidence that TLR4 signaling plays a key role in MSC transformation by inducing a pro-tumor phenotype associated with a permissive microenvironment allowing immune escape and tumor growth.

// Alessandra Romano 1 , Nunziatina Laura Parrinello 1 , Calogero Vetro 1 , Daniele Tibullo 1 , Cesarina Giallongo 1 , Piera La Cava 1 , Annalisa Chiarenza 1 , Giovanna Motta 1 , Anastasia L. Caruso 1 , Loredana Villari 2 , Claudio Tripodo 3 , Sebastiano Cosentino 4 , Massimo Ippolito 4 , Ugo Consoli 5 , Andrea Gallamini 6 , Stefano Pileri 7 , Francesco Di Raimondo 1 1 Division of Hematology, AOU “Policlinico - Vittorio Emanuele”, University of Catania, Catania, Italy 2 Division of Pathology, AOU “Policlinico - Vittorio Emanuele”, Catania, Italy 3 Tumor Immunology Unit, Department of Health Science, University of Palermo, Palermo, Italy 4 Nuclear Medicine Center, Azienda Ospedaliera Cannizzaro, Catania, Italy 5 Division of Hematology, ARNAS Garibaldi, Catania, Italy 6 Research, Innovation and Statistics Department. A. Lacassagne Cancer Centre, Nice, France 7 Unita di Diagnosi Emolinfopatologica, IEO, Milano, Italy Correspondence to: Alessandra Romano, email: sandrina.romano@gmail.com Francesco Di Raimondo, email: diraimon@unict.it Keywords: Arginase-1, Hodgkin lymphoma, PET-2 Received: June 30, 2016 Accepted: September 05, 2016 Published: September 14, 2016 ABSTRACT Purpose: Neutrophilia is hallmark of classic Hodgkin Lymphoma (cHL), but its precise characterization remains elusive. We aimed at investigating the immunosuppressive role of high-density neutrophils in HL. Experimental design: First, N-HL function was evaluated in vitro , showing increased arginase (Arg-1) expression and activity compared to healthy subjects. Second, we measured serum level of Arg-1 (s-Arg-1) by ELISA in two independent, training ( N = 40) and validation ( N = 78) sets. Results: s-Arg-1 was higher in patients with advanced stage ( p = 0.045), B-symptoms ( p = 0.0048) and a positive FDG-PET scan after two cycles of chemotherapy (PET-2, p = 0.012). Baseline levels of s-Arg-1 > 200 ng/mL resulted in 92% sensitivity and 56% specificity to predict a positive PET-2. Patients showing s-Arg-1 levels > 200 ng/mL had a shorter progression free survival (PFS). In multivariate analysis, PET-2 and s-Arg-1 at diagnosis were the only statistically significant prognostic variables related to PFS (respectively p = 0.0004 and p = 0.012). Moving from PET-2 status and s-Arg-1 level we constructed a prognostic score to predict long-term treatment outcome: low s-Arg-1 and negative PET-2 scan (score 0, N = 63), with a 3-Y PFS of 89.5%; either positive PET-2 or high s-Arg-1 (score 1, N = 46) with 3-Y PFS of 67.6%, and both positive markers (score 2, N = 9) with a 3-Y PFS of 37% ( p = 0.0004). Conclusions: We conclude that N-HL are immunosuppressive through increased Arg-1 expression, a novel potential biomarker for HL prognosis.

Introduction Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by hematopoietic/stem cell-derived clonal myeloproliferation leading to cytopenia/cytosis, splenomegaly and bone marrow (BM) fibrosis. The alteration of haematopoiesis associted with BM fibrosis is deeply associated with profound modifications of the BM microenvironment, as a consequence of a defective balance between the vascular niche and the endosteal niche, associated to megakaryocytes, endothelial and mesenchymal stromal cells (MSC) dysfunction through the production of a variety of profibrotic, angiogenic, and pro-inflammatory cytokines, including megakaryocyte-derived PDGF, TGF-beta and osteoprotegerin, IL-6, PDGF, RANTSs, BMP-2, and which can trigger auto-immune mechanisms, chronic inflammation and oxidative stress status. It is well-known that oncogenic lesions can switch the bioenergetics of malignant cells from OXPHOS to glycolysis (Warburg effect) with lactate production, a phenomenon clinically relevant, particularly in PMF where the amount of the enzyme lactate dehydrogenase (LDH) is an established biomarker of leucocyte turnover and an independent biomarker of overall and leukemia-free survival. The shuttling of the main LDH metabolite lactate is implicated in the interplay of cancer cells with neighboring stromal cells which become glycolytic and export lactate. In turn, lactate is taken up by cancer cells and used for oxidative metabolism, to drive angiogenesis in endothelial cells and to inhibit T- cell function. Moreover, there are evidences that lactate may be involved in the promotion of the fibrosis increasing the TGF-beta levels. In this work we aimed to investigate the role of lactate in the BM myelofibrosis. Results. From microarray datasets, we selected 34 PMF patients carrying the JAK2V617F mutation, 28 JAK2 wild-type patients and 16 healthy donors (HD). Our analysis showed that SLC16A1 (MCT1), SLC16A3(MCT2) and SLC16A7 (MCT4) genes were upregulated in patients in respect to healthy donors in a JAK2V617F mutation independent manner. Furthermore, we demonstrated a significant increase of lactate concentration in PB sera from PMF patients compared to HD, associated to higher percentage of circulating granulocyte- and monocyte-myeloid derived suppressor cells (Gr- and Mo-MDSCs), and Treg. Moreover, IDO, LAG3, BTLA, PDL-2, TIM-3 and CD152 levels were significantly increased in PMF sera compaterd to HD ones. To demonstrate that lactate could play a role in driving cancer immune evasion in PMF, healthy peripheral blood mononucleated cells (PBMCs) were incubated in presence of lactate. After 3 days we observed a significant increase of the percentage of Mo-MDSCs, Treg, CD4+PD1+ and CD8+PD1+ lymphocytes. The same results were obtained after incubation of PBMCs with sera from PMF patients. No effects were observed using HD sera. Interestingly, the percentages of Treg and Mo-MDSCs increased after exposure to PMF sera were significantly reduced in presence of the inhibitor of lactate transporter AZD3965. To investigate the role of lactate in the PMF microenvironment, we next exposed healthy MSCs to lactate for 48h. Treated MSCs assumed a CAF-like phenotype increasing expression of aSMA, FAP1 and TGF-beta. Moreover, Masson's trichrome staining showed an increase of collagen deposits in BM-MSCs associated to increased release of IL6, TGF-beta, MMP2, MMP9 and RANTES. Also, exposure to PMF sera induced higher collagen deposits in MSCs and this effect was reverted adding AZD3965. As BM fibrosis is frequently accompanied by osteosclerosis, we also investigated the effects of lactate on ostegenic differentiation of BM-MSCs. After 10 days of treatment with lactate, the BM-MSCs showed a morphological change associated to increased osteogenic gene markers such as BMP2, RUNX2 and SPARC (osteonectin), and higher released levels of calcitonin, BMP-2, MCP-1, sRANKL and osteoprotegerin. Conclusion Our results demonstrate that lactate might be involved in the immune impairment and BM fibrosis of PMF. The inhibition of lactate production and shuttles in myelofibrosis may be a strategy not only to inhibit invasive and metastatic behavior of cancer cells, but also to restore the anti-cancer immune response improving the results of therapy in MF patients. Disclosures Romano: Novartis: Honoraria; Takeda: Honoraria. Di Raimondo:Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen, Takeda, Novartis: Honoraria; Celgene: Consultancy, Honoraria; GILEAD, Incyte: Research Funding. Palumbo:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are rare hematological conditions known as myeloproliferative neoplasms (MPNs). They are characterized for being BCR-ABL negative malignancies and affected patients often present with symptoms which can significantly impact their quality of life. MPNs are characterized by a clonal proliferation of an abnormal hematopoietic stem/progenitor cell. In MPNs; cells of all myeloid lineages; including those involved in the immune and inflammatory response; may belong to the malignant clone thus leading to an altered immune response and an overexpression of cytokines and inflammatory receptors; further worsening chronic inflammation. Many of these cytokines; in particular, IL-1β and IL-18; are released in active form by activating the inflammasome complexes which in turn mediate the inflammatory process. Despite this; little is known about the functional effects of stem cell-driven inflammasome signaling in MPN pathogenesis. In this review we focused on the role of inflammatory pathway and inflammasome in MPN diseases. A better understanding of the inflammatory-state-driving MPNs and of the role of the inflammasome may provide new insights on possible therapeutic strategies

Background: Juvenile dermatomyositis (JDM) is a systemic, autoimmune, interferon (IFN)-mediated inflammatory muscle disorder that affects children younger than 18 years of age. JDM primarily affects the skin and the skeletal muscles. Interestingly, the role of viral infections has been hypothesized. Mammalian 2&prime, 5&prime, oligoadenylate synthetase (OAS) genes have been thoroughly characterized as components of the IFN-induced antiviral system, and they are connected to several innate immune-activated diseases. The main purpose of the paper is to define the potential interrelationship between the OAS gene family network and the molecular events that characterize JDM along with double-stranded RNA (dsRNA) molecular pathways. Methods: We analyzed three microarray datasets obtained from the NCBI in order to verify the expression levels of the OAS gene family network in muscle biopsies (MBx) of JDM patients compared to healthy controls. Furthermore, From GSE51392, we decided to select significant gene expression profiles of primary nasal and bronchial epithelial cells isolated from healthy subjects and treated with polyinosinic-polycytidylic acid (poly(I:C)), a synthetic analog of double-stranded RNA (dsRNA), a molecular pattern associated with viral infection. Results: The analysis showed that all OAS genes were modulated in JDM muscle biopsies. Furthermore, 99% of OASs gene family networks were significantly upregulated. Of importance, 39.9% of modulated genes in JDM overlapped with those of primary epithelial cells treated with poly(I:C). Moreover, the microarray analysis showed that the double-stranded dsRNA virus gene network was highly expressed. In addition, we showed that the innate/adaptive immunity markers were significantly expressed in JDM muscles biopsies. and that their levels were positively correlated to OAS gene family expression. Conclusion: OAS gene expression is extremely modulated in JDM as well as in the dsRNA viral gene network. These data lead us to speculate on the potential involvement of a viral infection as a trigger moment for this systemic autoimmune disease. Further in vitro and translational studies are needed to verify this hypothesis in order to strategically plan treatment interventions.

The chitotriosidase (CHIT-1) is a glycosyl hydrolase (GH), which has been found highly expressed in activated macrophages and in different monocyte-derived cell lines such as Kupffer cells and osteoclasts, as well is differently produced in diverse stages of macrophage polarization (M1 and M2). Recent finding suggests that CHIT-1 plays a crucial role in innate and acquired immunity. Dendritic cells (DCs) are a complex group of cells that play a critical role in immune response. The aim of this study was to investigate the presence of CHIT-1 during the differentiation and maturation of DCs. Magnetically-isolated peripheral blood monocytes were differentiated toward immature DCs (iDC) and mature DCs (mDCs). Our results showed, for the first time, that CHIT-1 is expressed during the process of differentiation and maturation of DCs in a time-dependent manner. We found that CHIT1 is evenly distributed in cytoplasm of both the iDCs and mDCs. Additionally, a significantly increased expression of CHIT1 mRNA and protein was observed in mature DCs. These results suggest that CHIT-1 play an important role in the DCs immunoresponse.

Significance: Several lines of evidence suggest that hematological malignancies exhibit an altered redox balance homeostasis that can lead to the activation of various survival pathways that, in turn, lead to the progression of disease and chemoresistance. Among these pathways, the heme oxygenase-1 (HO-1) pathway is likely to play a major role. HO catalyzes the enzymatic degradation of heme with the simultaneous release of carbon monoxide (CO), ferrous iron (Fe2+), and biliverdin. This review focuses on the role of HO-1 in various hematological malignancies and the possibility of exploiting such targets to improve the outcome of well-established chemotherapeutic regimens. Recent Advances and Critical Issues: Interestingly, the inhibition of the expression of HO-1 (e.g., with siRNA) or HO activity (with competitive inhibitors) contributes to the increased efficacy of chemotherapy and improves the outcome in animal models. Furthermore, some hematological malignancies (e.g., chronic myeloid leukemia...

BACKGROUND Multiple myeloma (MM) is a B-cell malignancy critically dependent for survival and proliferation on signals coming from its inflammatory microenvironment in which toll-like receptors (TLR) may be potential linking elements between inflammation and cancer. It has been recently demonstrated that TLR4 pathway provides a protective effect against bortezomib (BTZ)-induced endoplasmic reticulum (ER) stress and pre-treatment of MM cells with LPS significantly reduces BTZ-induced apoptosis. AIM Since the acquisition of BTZ resistance is accompanied by an increased reliance on mitochondrial respiration, we investigated the role of TLR4 as stress-responsive mechanism that protect mitochondria during BTZ-induced ER stress as potential mechanism of drug resistance. RESULTS The activation of TLR4 signaling by LPS increased mitochondrial mass in human MM cell lines (HMCL: U266, MM1.S, OPM2, NCI-H929) and induced up-regulation of mitochondrial biogenesis markers (PGC1a, PRC and TFAM). After treatment with BTZ for 24h, all HMCL over-expressed TLR4 and its signaling was functional as suggested by up-regulation of MyD88 and MAPK activation. Compared to BTZ-sensitive cells (U266-S), U266-R showed higher levels of TLR4, p-p38 and p-ERK proteins and higher mitochondrial mass. Using a selective TLR4 inhibitor (TAK-242), we next treated U266-R cells with either 15nM BTZ, 20 μM TAK-242 or their combination. Combinatorial treatment significantly induced cell apoptosis (about 52%; p CONCLUSION Taken together, these findings indicate thatTLR4 signaling is involved in the acquisition of bortezomib resistance protecting mitochondria during BTZ exposure and sustaining mitochondrial dynamics in BTZ-resitant cells. Inhibition of TLR4 may overcome bortezomib resistance in patients with relapsed/refractory MM. Disclosures Conticello: Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Palumbo:Celgene: Honoraria; Amgen: Honoraria; Hospira: Honoraria; Teva: Honoraria; Novartis: Honoraria; Janssen: Honoraria. Di Raimondo:Takeda: Consultancy; Amgen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by an accumulation of clonal plasma cells (PC) in the bone marrow (BM) leading to bone destruction and BM failure. Osteolytic bone disease is a common manifestation of MM that leads to progressive skeleton destruction and is the most severe cause of morbidity in MM patients. Pathogenetic mechanisms of MM bone destruction are closely linked to MM PC and osteoclasts (OCs) hyperactivity coupled with defective osteoblast (OB) function unable to counteract bone resorption. We recently demonstrated that the proteasome inhibitor (PI) Bortezomib, commonly used to treat MM, was capable to inhibit osteoclastic differentiation modulating chitinase family genes. In this work we investigated the effect of Ixazomib (IXA), a third generation PI, on osteoclastogenesis and osteogenic differentiation. Human monocytes were differentiated in OCs in presence of OC medium (supplemented with RANKL and M-CSF), and/or 10nM IXA. We observed that IXA was able to inhibit the expression of different OCs markers such as RANK, CTSK, TRAP, and MMP9 when added in OC medium in respect to OC medium alone (p Disclosures Conticello: Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Palumbo:Amgen: Honoraria; Celgene: Honoraria; Hospira: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Teva: Honoraria. Di Raimondo:Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy; Celgene: Consultancy, Honoraria, Research Funding.

In both monoclonal gammopathy of uncertain significance (MGUS) and multiple myeloma (MM) patients, immune functions are variably impaired, and there is a high risk of bacterial infections. Neutrophils are the most abundant circulating leukocytes and constitute the first line of host defense. Since little is known about the contribution of autophagy in the neutrophil function of MGUS and MM patients, we investigated the basal autophagy flux in freshly sorted neutrophils of patients and tested the plastic response of healthy neutrophils to soluble factors of MM. In freshly sorted high-density neutrophils obtained from patients with MGUS and MM or healthy subjects, we found a progressive autophagy trigger associated with soluble factors circulating in both peripheral blood and bone marrow, associated with increased IFN&gamma, and pSTAT3S727. In normal high-density neutrophils, the formation of acidic vesicular organelles, a morphological characteristic of autophagy, could be induced after exposure for three hours with myeloma conditioned media or MM sera, an effect associated with increased phosphorylation of STAT3-pS727 and reverted by treatment with pan-JAK2 inhibitor ruxolitinib. Taken together, our data suggest that soluble factors in MM can trigger contemporary JAK2 signaling and autophagy in neutrophils, targetable with ruxolitinib.

The molecular nature of lipoic acid (LA) clarifies its capability of taking part to a variety of biochemical reactions where redox state is meaningful. The pivotal action of LA is the antioxidant activity due to its ability to scavenge and inactivate free radicals. Furthermore, LA has been shown to chelate toxic metals both directly and indirectly by its capability to enhance intracellular glutathione (GSH) levels. This last property is due to its ability to interact with GSH and recycle endogenous GSH. LA exhibits significant antioxidant activity protecting against oxidative damage in several diseases, including neurodegenerative disorders. Interestingly, LA is unique among natural antioxidants for its capability to satisfy a lot of requirements, making it a potentially highly effective therapeutic agent for many conditions related with oxidative damage. In particular, there are evidences showing that LA has therapeutic activity in lowering glucose levels in diabetic conditions. Similarly, LA supplementation has multiple beneficial effects on the regression of the mitochondrial function and on oxidative stress associated with several diseases and aging. The aim of the present review is to describe the molecular mechanisms underlying the beneficial effects of LA under various experimental conditions and disease and how to exploit such effect for clinical purposes. LA has pleiotropic effects in different pathways related with several diseases, its use as a potential therapeutic agent is very promising.

Azacytidine (5-AZA) is the standard first-choice treatment for high-risk myelodysplasia (MDS) patients. However, the clinical outcome for those patients who interrupt treatment or whose disease failed to respond is very poor. In order to identify the cellular pathways that are modified by long-term exposure to 5-AZA, we evaluated key proteins associated with the autophagy pathway by reverse-phase microarray (RPPA). Comparing bone marrow mononucleated cells (BMMCs) obtained from 20 newly-diagnosed patients and after four 5-AZA cycles we found an increased autophagy signaling. We then evaluated ex-vivo the effect of the combination of 5-AZA with autophagy inhibitors chloroquine (CQ) and leupeptin. Since 5-AZA and CQ showed synergism due to an increase of basal autophagy after 5-AZA exposure, we adopted a sequential treatment treating BMMCs with 5 μM 5-AZA for 72 h followed by 10 μM CQ for 24 h and found increased apoptosis, associated to a reduction of G2M phase and increase in G0-G1 phase. Long-term exposure to 5-AZA induced the reduction of the autophagic marker SQSTM1/p62, reversible by CQ or leupeptin exposure. In conclusion, we identified autophagy as a compensatory pathway occurring in MDS-BM after long-term exposure to 5-AZA and we provided evidences that a sequential treatment of 5-AZA followed by CQ could improve 5-AZA efficacy, providing novel insight for tailored therapy in MDS patients progressing after 5-AZA therapy.

The chimeric BCR⁄ABL gene, generated by a t(9;22)chromosome translocation (de Klein, et al. 1982) is thehallmark of chronic myeloid leukemia (CML) (Majstereket al., 2006a). The fusion oncogene encodes a proteintyrosine kinase, constitutively activated, that facilitatesthe expansion of the neoplastic clone (Ren 2005).BCR⁄ABL tyrosine kinase allows cells to proliferate inthe absence of growth factors, protects them from apop-tosis and renders cells resistant to genotoxic treatment(Canitrot, et al. 2003).TheCMLcellsaremoreresistanttoconventionalchemo-therapy than other leukemic cells and combination chemo-therapy is able to induce only a transient reduction of theleukemic burden but it is notable to induce atrue completeremission (Kantarjian, et al. 1985). Even high doses of che-motherapyfollowedbyautologousstemcellsupport(Buttu-rini, et al. 1990) or syngeneic bone marrow transplantation(GaleandButturini1994)failtocureCMLpatients.Allthesedata support the concept that CML cells are resistant toapoptosis induced by chemotherapy. Several mechanismsAbstractBCR⁄ABL positive cells are known to be resistant to DNA damage induced by chemotherapy while theyare sensitive to imatinib (IM), a tyrosine kinase inhibitor (TKI). To evaluate whether this drug can increasethe activity of cytotoxic drugs on BCR⁄ABL positive cells, we measured the toxicity of cytosine arabinoside(ARA-C), hydroxyurea (HU) and melphalan (MEL), after a pretreatment of 24 h with IM on K562 cell line.The highest cytotoxic effect was seen when the TKI was followed by MEL; our results indicate that inhibi-tion of BCR⁄ABL activity by IM increased the cytotoxicity of MEL by favoring the DNA damage induced bythis drug and by shortening the time for DNA repair at the G2⁄M checkpoint. A stronger activation ofsome genes involved in both intrinsic and extrinsic apoptotic pathways was also observed with IM⁄MELcombination compared to IM or MEL alone. The drugs association was further tested in a type of BaF3cells (TonB.210) where the BCR-ABL expression is inducible by doxycycline; in this model it was con-firmed that a reduction of BCR⁄ABL activity resulted in an increased susceptibility to the cytotoxic effectof MEL. Furthermore, we studied the effect of IM⁄MEL treatment on the proliferative potential of myeloidprogenitors of six CML patients at diagnosis. The analysis of CFU-GM and BFU-E colonies demonstratedthat the IM⁄MEL combination was more effective than IM alone in reducing the overall number of colo-nies and the number of copies of BCR⁄ABL. In conclusion, our work shows that inhibition of BCR⁄ABLactivity increases the toxicity of MEL and allows an efficient killing of leukemic cells, suggesting that a clin-ical development of this approach could have therapeutic advantages for CML patients.

Background and Objective : There is an increasing interest about the role of amino acid degrading enzymes in cancer immunotherapy.In multiple myeloma (MM) disease progression depends on the ability of malignant plasma cells (PC) to subvert the local microenvironment and reshape host immunity to support tumor growth. Our previous work showed that i) MM cells have metabolic plasticity due to availability of external nutrients; ii) aminoacid shortage due to the tryptohan (trp) degrading enzyme IDO-1 was associated to clinical MM progression. Thus, we tested bio-energetic changes in MM upon trp deprivation. Experimental design : We evaluated the trascriptome changes in the adaptive response in four human MM cell lines (MM1.s, H929, U266, OPM2), chosen for their cytogenetic alterations and previously characterized for differential expression of CD38 and c-myc and primary BM samples of MGUS and MM to trp shortage. The pathway was confirmed looked at the expression levels of key metabolic genes in a large series of highly purified BM PC samples from healthy donors (4N), 129 MM, 24 primary plasma cell leukemia (pPCL), 12 secondary PCL (sPCL) cases from a proprietary dataset (GSE66293). Results Trp deprivation induced an adaptive response through increased expression, time and dose-dependent, of ATF4-ASNS-CHOP-GADD34, part of GCN2 signaling, associated to increased expression of p62 (the main autophagic receptor in MM) followed by autophagy flux induction, as shown by flow cytometry and immunofluorescence evaluations. In MM cell lines, trp deprivation altered the cellular dependence on mitochondrial ATP generation via oxidative phosphorylation, inducing in vitro increase of lactate dehydrogenase (LDH) and intracellular lactate and NAD/NADH with down-regulation of surface expression of the NAD-ase CD38. Similarly, short-term trp deprivation in CD138+ plasma cells isolated by magnetic sorting from MM patients, downregulated surface expression of CD38 and intra-cellular amount of c-myc, as detected by flow cytometry. This phenomenon was associated to increased isocitrate dehydrogenase (IDH-1), enolase-1 (ENO-1), phosphoglycerate kinase 1 (PGK-1), and dihydrolipoamide dehydrogenase (DLD), suggesting that branched chain amino acids with tolerogenic meaning like trp are used in MM to sustain NADH availability and energy production. In the GSE66293 MM progression was associated to increased expression of ENO-1, PGK-1, and DLD, suggesting that changes in transcription of genes involved in bioenergetics could be clinically relevant. Conclusion Overall, our study reveals that trp deprivation promotes an adaptive response of MM offering new putative targets in the bio-energetic pathway for synthetic lethality. Disclosures Palumbo: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Di Raimondo:Takeda: Honoraria, Research Funding; Celgene: Honoraria.

It is well known that mesenchymal stem cells (MSC) have a role in promotion of tumor growth, survival and drug-resistance in chronic myeloid leukemia (CML). Recent reports indicated that a subpopulation of myeloid cells, defined as granulocyte-like myeloid-derived suppressor cells (G-MDSC) is increased in these patients. So far, the role of MSC in MDSC expansion and activation into the BM microenvironment remains unexplored. To address this question, here we use a specific experimental model in vitro, co-culturing MSC with peripheral blood mononucleated cells (PBMC) from normal individuals, in order to generate MSC-educated G-MDSC. Although MSC of healthy donors (HD) and CML patients were able to generate the same amount of MDSC, only CML-MSC-educated G-MDSC exhibited suppressive ability on autologous T lymphocytes. In addition, compared with HD-MSC, CML-MSC over-expressed some immunomodulatory factors including TGFβ, IL6 and IL10, that could be involved in MDSC activation. CML-MSC-educated G-MDSC expressed higher levels of ARG1, TNFα, IL1β, COX2 and IL6 than G-MDSC isolated from co-culture with HD-MSC. Our data provide evidence that CML-MSC may play a critical role in tumor microenvironment by orchestrating G-MDSC activation and regulating T lymphocytes-mediated leukemia surveillance, thus contributing to CML immune escape.

Granulocytic-Myeloid-derived suppressor cells (G-MDSC) are increased in Multiple Myeloma (MM) patients but the mechanisms of G-MDSC generation are still unknown. There are many evidences of the role of mesenchymal stem cells (MSC) in promoting MM cell growth, survival and drug-resistance. We here used a specific experimental model in vitro to evaluate the ability of MSC to induce G-MDSC. We found that although MSC derived from healthy donors (HD), MGUS and MM were able to generate the same amount of MDSC, only MM-MSC-educated G-MDSC exhibited suppressive ability. In addition, in comparison with MSC derived from HD, MM-MSC produce higher amount of immune-modulatory factors that could be involved in MDSC induction. Compared to G-MDSC obtained from co-culture models with MSC from healthy subjects, both MGUS and MM-MSC-educated G-MDSC showed increase of immune-modulatory factors. However, only MM-MSC educated G-MDSC 1) up-regulated immune-suppressive factors as ARG1 and TNFα, 2) expressed higher levels of PROK2, important in angiogenesis and inflammatory process, and 3) showed ability to digest bone matrix. Our data demonstrate that MM-MSC are functionally different from healthy subjects and MGUS-MSC, supporting an evolving concept regarding the contribution of MM-MSC to tumor development and progression.

We analyzed FcgammaRIIIA-158V/F and FcgammaRIIA-131H/R polymorphisms in a cohort of 94 newly diagnosed follicular lymphoma (FL) patients sequentially treated with CHOP and Rituximab. With a median follow-up of 5.8 years, the overall survival at 8 years is 83%. Univariate and multivariate analysis showed no correlation between FcgammaRIIIA-158VV/VF and FcgammaRIIA-131HH/HR polymorphisms and the overall response rate, the molecular response and the event-free survival obtained after CHOP and Rituximab. By contrast, the achievement of a durable molecular clearance of BCL2/IgH+ cells detectable in the bone marrow is confirmed to be a reliable predictive factor of a better long-term clinical outcome.

Mantle cell lymphoma (MCL) accounts for 3-10% of all non-Hodgkin's lymphomas, with median overall survival not exceeding 3-4 years. Rituximab in combination with the Hyper-CVAD regimen appears the most promising regimen; thus, we adopted it as a first-line treatment strategy in a series of 24 patients. In addition to evaluation of clinical success of the regimen, we investigated a possible role of polymorphism in IgG Fc receptors, FCgammaRIIIa and FCgammaRIIa. The frequencies of FCgammaRIIIa-158 were as follows: V/V=4/24 (17%); V/F=16/24 (66%); F/F=4/24 (17%). Those of the FCgammaRIIa-131 polymorphism were H/H=11/24 (46%), H/R=9/24 (37%), R/R=4/24 (17%). The overall response rate was 62.5%, with 33% of complete responses (CRs) after four cycles of R-Hyper-CVAD. Two-year progression-free survival (PFS) was 78% for 158V/V patients vs 75% for cases carrying phenylalanine (p=0.88). When the FCgammaRIIa polymorphism was assessed, the 2-year PFS was 82% for 131H/H patients vs 75% for those carrying arginine (p=0.26). Eighty-three percent of cases achieved Polymerase Chain Reaction (PCR)-negativity: the progression rate was significantly influenced by the minimal residual disease clearance, with 12% progression in the subgroup of PCR-negative cases versus 67% progression in PCR-positive cases (p=0.008). The achievement of PCRnegativity was not significantly influenced by FCgammaR polymorphisms. Results confirm that rituximab plus Hyper-CVAD is an effective regimen for the induction of prolonged remission in patients with aggressive MCL and suggest that rituximab efficacy is independent of the FCgammaR polymorphisms.

The suppression of the immune system creates a permissive environment for development and progression of cancer. One population of immunosuppressive cells that have become the focus of intense study is myeloid derived suppressor cells (MDSCs), immature myeloid cells able to induce immune-escape, angiogenesis, and tumor progression. Two different subpopulations have been identified and studied: granulocytic and monocytic MDSCs, with a different immunophenotype and immunosuppressive properties. Recently, an accumulation of both Gr-MDSCs and Mo-MDSCs cells has been found in the peripheral blood of chronic myeloid leukemia (CML) patients. They are part of the tumor clone showing BCR/ABL expression. Imatinib therapy decreases both MDSCs and arginase 1 levels to normal ones. This review will focus on actual knowledge for human MDSCs and their immunosuppressive activity in CML patients, with a critical attention to comparison of Gr-MDSCs and polymorphonuclear cells (PMNs). We will then suggest the monitoring of MDSCs in patients who have discontinued tyrosine kinase inhibitors (TKIs) therapy to evaluate if their increase could correlate with disease relapse.

Chitinase-3-like-1 protein (CHI3L1) is a glycosyl hydrolase (GH) highly expressed in a variety of inflammatory diseases at infectious and non-infectious etiology. CHI3L1 is produced by a wide variety of cells including monocyte-derived macrophages cell lines such as polarized M1 and M2 type macrophages, osteoclasts and Kupffer cells. In this study we have examined the expression of CHI3L1 during the differentiation and maturation of dendritic cells. Magnetically-isolated peripheral blood monocytes were differentiated toward immature DCs (iDC) and mature DCs (mDCs) through a combination of factors and cytokines. Our result showed, for the first time, that CHI3L1 is expressed during the process of differentiation and maturation of dendritic cells in time dependent manner. Furthermore, the CHI3L1 is evenly distributed in cytoplasm and in the nucleus of both the iDCs and mDCs. These results suggest that CHI3L1 may play crucial role in the DCs immunoresponse.

INTRODUCTION. The complex interplay between cancer cells and immune system allows neoplastic cells to evade immune surveillance and expand. Recently, our and another group have demonstrated that a subpopulation of myeloid cells, defined as "granulocytic myeloid-derived suppressor cells" (G-MDSC), plays an important role for immune escape in chronic myeloid leukemia (CML) patients by reducing T cell activation. The aim of this study was to evaluate the influence of Mesenchymal stem cells (MSC) on generation of MDSCs by comparing CML MSCs (n=10) with healthy donors (HD) MSC (n=8). METHODS. G-MDSC (CD11b+CD33+CD14-HLADR- cells) were analyzed in peripheral blood (PB) of 20 healthy donors (HD) and 30 CML patients at diagnosis by cytofluorimetric analysis. Immuno-suppressive activity was tested through incubation of G-MDSC with autologous CFSE-labeled T cells and stimulation with phytohaemagglutinin (PHA). Controls included a positive T cell proliferation control (T cells plus PHA) and a negative one (T cells only). After three days, T cell proliferation was analyzed by flow cytometry. For G-MDSC generation, human peripheral blood mononucleated cells (PBMC) from HD were cultured alone and with MSC of CML (n=10) or HD (n=8) (1:100 ratio). After one week, G-MDSC were isolated using anti-CD66b magnetic microbeads and the phenotype was confirmed by cytofluorimetric analysis. Expression of ARG1, NOS2, PTGS2, TNFα, TGFβ, IL6, IL10, IL1β was also evaluated using real time PCR. RESULTS. Percentage of cells with a G-MDSC phenotype was greater in PB obtained from CML patients than HD (82.5±9.6% vs 56,2±5.4%, p CONCLUSION. Our work demonstrates that CML MSCs are able to activate MDSCs favoring cancer immune evasion in CML patients. Disclosures Palumbo: Novartis: Honoraria, Other: Advisory Board.

Mammalian chitinases consisting of CHIA, CHIT1, CHI3L1, CHI3L2 and CHID1 exert important biological roles in the monocyte lineage and chronic inflammatory diseases. Pathological bone resorption is a cause of significant morbidity in diseases affecting the skeleton such as rheumatoid arthritis, osteoporosis, periodontitis and cancer metastasis. The biologic role of chitinases in bone resorption is poorly understood. In this study, we evaluated the expression of the chitinases family during osteoclast differentiation. The expression of CHIA, CHI3L2 and CHID1 resulted unchanged during osteoclast differentiation, whereas CHIT1 and CHI3L1 increased significantly. We also observed that CHIT1 and CHI3L1 are involved in osteoclast function. Indeed, silencing CHIT1 and CHI3L1 with siRNA resulted in a significant decrease in bone resorption activity. In addition, transfection with CHIT1 or CHI3L1 siRNA and co-transfection with both decreased the levels of the pro-differentiative marker MMP9. Overall, these discoveries reveal a novel and crucial role for both CHIT1 and CHI3L1 in promoting bone resorption and identifying new potential candidate markers for therapeutic targeting.

Tumor immune tolerance can derive from the recruitment of suppressor cell population, including myeloid derived suppressor cells (MDSCs), able to inhibit T cells activity. We identified a significantly expanded MDSCs population in chronic myeloid leukemia (CML) patients at diagnosis that decreased to normal levels after imatinib therapy. In addition, expression of arginase 1 (Arg1) that depletes microenvironment of arginine, an essential aminoacid for T cell function, resulted in an increase in patients at diagnosis. Purified CML CD11b+CD33+CD14-HLADR- cells markedly suppressed normal donor T cell proliferation in vitro. Comparing CML Gr-MDSCs to autologous polymorphonuclear leukocytes (PMNs) we observed a higher Arg1 expression and activity in PMNs, together with an inhibitory effect on T cells in vitro. Our data indicate that CML cells create an immuno-tolerant environment associated to MDSCs expansion with immunosuppressive capacity mediated by Arg1. In addition, we demonstrated for the first time also an immunosuppressive activity of CML PMNs, suggesting a strong potential immune escape mechanism created by CML cells, which control the anti-tumor reactive T cells. MDSCs should be monitored in imatinib discontinuation trials to understand their importance in relapsing patients.

The recent advances in cytogenetic and molecular diagnostic techniques has provided a better understanding of biology of CLL and a better prediction of disease progression and refractoriness but they are not easily accessible to all Institutions. CLL cells depend on microenvironmental interactions for proliferation and survival that are at least partially mediated through B cell receptor (BCR) signaling. An increased number of studies describe the emerging role of neutrophils and monocytes as mediators of the inflammation process and antitumor immunity modulation that support tumorigenesis and tumor progression. The neutrophil to lymphocyte ratio (NLR), has been recently published as new marker of systemic inflammation associated with outcome in different cancer types. In our study we retrospectively evaluated the prognostic significance of peripheral blood neutrophils, monocytes and non-neoplastic lymphocytes in a unicentric, unselected, CLL cohort of 400 CLL patients (160 were treated). The study was approved by the institutional board review;informed consent was obtained from patients. Using the blood count and the Flow cytometric analysis reports, peripheral blood absolute neutrophil count (ANC), absolute monocyte count (AMC) and absolute T-lymphocyte count (ALC-CD3+) were evaluated and the neutrophil to T-lymphocyte ratio (NLR), the monocyte to T-lymphocyte ratio (MLR) and the neutrophil to monocyte ratio (NMR) were calculated. Clinical and biological data from all patients were also retrieved. Serial count and ratio were evaluated at diagnosis, during follow up and at relapse to make a better comparison with the major clinical and prognostic markers commonly used. The median ratio for NLR 2.67 and patients with pre-treatment NLR > 3 had a shorter time from diagnosis to treatment initiation. CLL patients showed an increase in the absolute number of monocytes compared to normal controls (788±65 cells/μL vs 469±51 cells/μL, p=0.005) and MLR appeared higher in advanced stage (stage Binet C) and bulky disease (p=0.06). High level of both NLR and MLR (cut off >3) correlated with the presence of a complex karyotype detected by FISH (p=0.03, p=0.016). NLR ratio was associated with the absence of serum prognostic markers, such as the expression of CD38, ZAP-70 and CD49d. This result, apparently conflicting, could strengthen the NLR as an independent prognostic factor. NMR was higher in asymptomatic patients (absence vs. presence of B symptoms, p=0.02) and this data resulted independent from disease stage. NMR median value was significantly higher in untreated patients than in patients who received treatment (p=0.01), strengthening the hypothesis that this ratio is associated with a more indolent form of disease. In this contest, the subset of patients with CD49d positive disease showed the lowest NMR value. This data seems of relevance, being CD49d a recently discovered and validated prognostic marker. ANC/ALC and AMC/ALC ratio significantly increased at relapse compare to baseline (NLR average onset 2.3±0.4 vs 3.4±0.6 at relapse). The median NMR value showed conversely the opposite trend: NMR is reduced in first relapse (NMR average onset 7.2±0.4 vs. 5.4±0.5 at relapse). These combined ratios reflect both the inflammatory status, the immune response and the microenviromental network that contribute to aggressive tumor biology and disease progression. They are simple, cheap, easily measured and reproducible and can be integrated into daily clinical practice as new prognostic markers for CLL. Disclosures Chiarenza: Gilead: Consultancy; Janssen: Consultancy; Roche: Consultancy.

Background and Objective : Our previous work showed that neutrophils sedimenting on top of red cells after density gradients, defined as high-density neutrophils (HDN) are emerging as pro-tumoral players with immunesuppressive properties, by depleting the surrounding microenvironment from arginine (arg) and tryptophan (trp) through increased expression of arginase (Arg-1) and 2-3 indoleamine deoxygenase (IDO), but little is known about their clinical relevance in Multiple Myeloma (MM). Experimental design : We integrated two distinct unbiased approaches. First, we assessed gene expression profiles (GEP) and functional evaluation of HDN sorted from 60 newly diagnosed MM patients, 30 patients with MGUS, and 30 healthy subjects.. Second, we adopted ultra-high performance liquid and gas chromatography followed by mass spectrometry (UHPLC/GC-MS) on an independent series of 167 samples of bone marrow (BM) and peripheral plasma collected ad hocfrom 125 individuals, comprising newly diagnosed MM, MGUS, smoldering MM, and age-matched healthy volunteers (n=29). Progression-free survival in newly diagnosed MM patients was correlated respectively to HDN functional and expression features or metabolic profiles in the two series. Results : GEP analyses disclosed that, compared to MGUS and HD, MM-HDN showed increased expression of Arg-1, IDO-1 and the angiogenic factor PROK-2/BV8 , confirmed by qRT-PCR and immunofluorescence. Indeed, MM-HDN (but not MGUS- and HD-HDN) revealed immunosuppressive activity in co-cultures with allogeneic T-cells, reverted by the selective Arg-1 inhibitor nor-NOHA. In addition, circulating Arg-1 in serum was higher in MM (169.5 ± 17.6) than MGUS (93.2 ± 10.4, p=0.0028) and healthy subjects (70.1 ± 16.5 ng/ml, p=0.0017). In MM patients there was a progressive increase from ISS stage I through III (p=0.014). High Arg-1 in MM patients' sera at diagnosis predicted clinical outcome after first line therapy, being associated to shorter progression-free survival (respectively 22.4 months versus unreached median, p=0.01). Metabolic profiling independently identified the same immunosuppressive Arg-1-urea-arginine (arg) and tryptophan (trp)-kinurenine-IDO pathways, with arg and trp significantly reduced along MM evolution. Lower levels of arg were associated to shorter PFS (37.2 versus 17.6 months, p=0.023). We then tested if aminoacid depletion (arg and trp) favors PCs growth. When exposed to trp-free or arg-free medium, human MM cell lines expressed more CHOP, p62, m-TOR and Blimp-1 (markers respectively of autophagy induction and immunoglobulin production); conversely, p62 silencing reduced Blimp-1, confirming the important role of p62-dependent mechanism in supporting PCs fitness. Conclusion : In MM, HDN are immunosuppressive due to increased Arg-1 and IDO expression. We propose the arginine/Arg-1 axis as a novel potential biomarker for MM prognosis. Disclosures Ciceri: MolMed SpA: Consultancy.

Purpose : Neutrophilia is hallmark of classic Hodgkin Lymphoma (cHL), but its precise characterization remains elusive. We aimed at investigating the immunosuppressive role of high-density neutrophils in HL. Experimental design : First, N-HL function was evaluated in vitro, showing increased arginase (Arg-1) expression and activity compared to healthy subjects. Second, we measured serum level of Arg-1 (s-Arg-1) by ELISA in two independent, training (N=40) and validation (N=78) sets. Results : s-Arg-1 was higher in patients with advanced stage (p=0.045), B-symptoms (p=0.0048) and a positive FDG-PET scan after two cycles of chemotherapy (PET-2, p=0.012). Baseline levels of s-Arg-1 >200 ng/mL resulted in 92% sensitivity and 56% specificity to predict a positive PET-2. Patients showing s-Arg-1 levels > 200 ng/mL had a shorter progression free survival (PFS). In multivariate analysis, PET-2 and s-Arg-1 at diagnosis were the only statistically significant prognostic variables related to PFS (respectively p=0.0004 and p=0.012). Moving from PET-2 status and s-Arg-1 level we constructed a prognostic score to predict long-term treatment outcome: low s-Arg-1 and negative PET-2 scan (score 0, N=63), with a 3-Y PFS of 89.5%; either positive PET-2 or high s-Arg-1 (score 1, N=46) with 3-Y PFS of 67.6%, and both positive markers (score 2, N=9) with a 3-Y PFS of 37% (p=0.0004). Conclusions : We conclude that N-HL are immunosuppressive through increased Arg-1 expression, a potential novel potential biomarker for HL prognosis. Disclosures Vetro: MLL Munich Leukemia Laboratory: Employment. Chiarenza:Gilead: Consultancy; Janssen: Consultancy; Roche: Consultancy.

Background and objective: Multiple Myeloma (MM) is a plasma cell malignancy with a well documented immune dysfunction. Previous reports in murine models and our previous work in MM patients, showed that myeloid cells, both granulocyte-myeloid derived suppressor cells (G-MDSC) and neutrophils sedimenting on top of red cells after density gradients, defined as high-density neutrophils (HDN) are increased and exert an immunosuppressive activity. Moreover, we showed that myeloid precursors could be activate and acquire G-MDSC phenotype if cultures in presence of MM mesenchymal cells. Thus, we characterized functionally HDN to gauge their contribution in immunesuppression, angiogenesis and bone resorption in MM microenvironment. Methods: In HDN freshly-isolated from a series of 60 newly-diagnosed MM, 30 smoldering MM and 30 MGUS we investigated by RT-PCR, flow-cytometry, western-blot and immunofluorescence genes and proteins related to immunesuppression (Arg-1, IDO), angiogenesis (BV8, VEGF, MMP9) and bone resorption (IL1-b, MMP9, CHI3L). Results: First, MM-HDN exhibited an increased expression of immunesuppressive molecules Arg-1 and IDO compared to MGUS and healthy subjects (25.5 vs 6.2 vs 1 fold changes in gene expression, p=0.003), confirmed by functional assay of enzymatic activity, immunostaining and WB, positively correlated with advanced disease. Second, MM-HDN showed increased expression of BV8 and MMP9 (p=0.01, p=0.05) but not VEGF, as confirmed by immunofluorescence. In line with this observation, by using migration assay (CIM plate) MM-HDN induced neo-vasculogenesis in vitro by forming more and aberrant tubes than healthy or MGUS-HDN, thus to disclosure a novel proangiogenic mechanism driven by BV8. Third, MM-HDN showed increased expression of IL1-b, MMP9, CHI3L, associated to increased bone resorption activity, evaluated by dentin-disk assay. The same findings could be obtained treating bone matrix with MM-HDN-conditioned media, suggesting the crucial role of molecules released by MM-HDN but no from MGUS-HDN. Conclusion: taken together, our observations disclosure the contribution of aberrantly activated HDN in MM clinical features, contributing to immunosuppression, angiogenesis and bone resorption. Disclosures No relevant conflicts of interest to declare.

Purpose A well-recognized feature of MM is the intimate relationship between plasma cells and bone marrow microenvironment, which is mainly composed of MSC, endothelial cells, immune cells and extracellular matrix. G-MDSC accumulate in the tumor microenvironment during tumor development promoting tumor growth and immunosuppression. Aim Analyzing MSC from MGUS, Smoldering myeloma (SMM) and MM patients in promoting G-MDSC generation. Methods Human peripheral blood mononucleated cells (PBMC) isolated from healthy subjects (HS) were cultured alone and with HS- (n=10), MGUS- (n=10), SMM- (n=4) or MM-MSC (n=14)(1:100). After 6 days, G-MDSC were isolated using anti-CD66b magnetic microbeads and the phenotype(CD11b+CD33+CD14-HLADR-) was confirmed by cytofluorimetric analysis. Results Only G-MDSC educated by SMM- and MM-MSC co-cultures (MSCed-G-MDSC) were able to suppress T cell proliferation when cultured with normal lymphocytes (p Adding Bortezomib (5 nM), Lenalidomide (10 μM) or Pomalidomide (1 nM) during co-culture with MM-MSC, isolated G-MDSC showed a significant reduction of pro-angiogenic and bone resorption activity (p Conclusion MSC play a key role promoting tumor microenvironment transformation in SMM and MM patients.Indeed, only SMM- and MM-MSC and not MGUS-MSC are able to activate myeloid cells in G-MDSC with immunosuppressive, pro-angiogenic, and bone resorptionactivity. Disclosures No relevant conflicts of interest to declare.

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by an accumulation of clonal plasma cells (PC) in the bone marrow (BM) leading to bone destruction and BM failure. Despite recent advances in pharmacological therapy, MM remains a largely incurable pathology. Therefore, novel effective and less toxic agents are urgently necessary. In the last few years, pomegranate has been studied for its potential therapeutic properties including treatment and prevention of cancer. Pomegranate juice (PGJ) contains a number of potential active compounds including organic acids, vitamins, sugars, and phenolic components that are all responsible of the pro-apoptotic effects observed in tumor cell line. The aim of present investigation is to assess the antiproliferative and antiangiogenic potential of the PGJ in human multiple myeloma cell lines. Our data demonstrate the anti-proliferative potential of PGJ in MM cells; its ability to induce G0/G1 cell cycle block and its anti-angiogenic effects. Interestingly, sequential combination of bortezomib/PGJ improved the cytotoxic effect of the proteosome inhibitor. We investigated the effect of PGJ on angiogenesis and cell migration/invasion. Interestingly, we observed an inhibitory effect on the tube formation, microvessel outgrowth aorting ring and decreased cell migration and invasion as showed by wound-healing and transwell assays, respectively. Analysis of angiogenic genes expression in endothelial cells confirmed the anti-angiogenic properties of pomegranate. Therefore, PGJ administration could represent a good tool in order to identify novel therapeutic strategies for MM treatment, exploiting its anti-proliferative and anti-angiogenic effects. Finally, the present research supports the evidence that PGJ could play a key role of a future therapeutic approach for treatment of MM in order to optimize the pharmacological effect of bortezomib, especially as adjuvant after treatment.

Abstract 3386 It has been demonstrated that long term treatment of CML patients with Imatinib (IM) is associated with altered bone and mineral metabolism. The mechanisms that are responsible for this effect are not fully understood but an inhibition of the PDGF-Rβ (Platelet Derived Growth Factor- Receptor beta) axis has been documented. In order to evaluate if stimulation of osteoblastogenesis is a common feature of other tyrosine kinase inhibitors (TKIs) approved for the treatment of patients with CML, we evaluated the osteoblatic differentiation of Mesenchymal Stem Cells derived from adult bone marrow donors (BM-MSCs) after in vitro treatment with Dasatinib (DA), Nilotinib (NI) or Bosutinib (BO). BM-MSCs were induced to differentiate in osteoblastic cells by treatment with osteogenic medium (OM) with or without DA, NI or BO. BM-MSCs was induced to differentiate in osteoblastic cells by treatmet with osteogenic medium (0.2 mM ascorbic acid, 0.1 μ m dexamethasone and 10 mM β-glycerophosphate, OM) with or without DA 2nM, BO 5nM or NI 100nM. After 21 days of treatment, in the cultures treated with DA e NI for 21 days we have observed a significant increase of extracellular mineralization and of osteogenic markers such as bone morphogenetic protein (BMP2) (p In conclusion, we show that besides IM, other TKIs such as DA and NI, but not BO, increase osteogenic markers in hBM-MSCs. Since BO differs from the other TKIs because of its low affinity to PDGF-R, these experiments suggest that inhibition of PDGF-R is a major pathway in the induction of osteoblastogenesis by TKIs. In addition, a perturbation of bone metabolism induced by TKi may interfere with physiological growth and have negative consequences in children and adolescents. In this subset of patients, treatment with BO could became the first choice because we have demonstrated that this drug does not affect osteoblastogenesis. Disclosures: No relevant conflicts of interest to declare.

The advent of biological targeted therapies has changed the prognosis of several hematologic and solid malignancies including Chronic Myeloid Leukemia (CML), breast, colo-rectal (CRC) and head and ...

Both arsenic trioxide (ATO) and bortezomib show separate antileukemic activity. With the purpose of evaluating whether the combination of ATO and bortezomib would be an option for patients with acute leukemia, we incubated HL60 leukemic cells with ATO alone and in combination with bortezomib. ATO and bortezomib cooperated to induce cell death and to inhibit proliferation and apoptosis in a synergistic way. The combined treatment resulted in a stronger activation of caspase 8 and 9, moderate activation of caspase 3, and increased expression of Fas and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-DR5 receptors. When bortezomib was added, some proapoptotic genes (CARD9, TRAIL) were upregulated, and some antiapoptotic genes (BCL2, BCL3, FLICE) were downregulated. When coincubated, approximately 80% of cells showed altered mitochondrial membrane permeability. Moreover, ATO alone and in combination with bortezomib abrogated DNA-binding activity of nuclear factor kappa beta (NF-kappaB). Gene expression assays showed that more deregulated genes were related to proliferation of leukocytes, tumorigenesis, control of cell cycle, hypoxia and oxidative stress, cytokines, PI3K-AKT, ERK-MAPK, EGF pathways, and ubiquitination. Finally, in three cases of acute myeloid leukemia, the addition of bortezomib to ATO significantly increased cytotoxicity. We conclude that the combination of bortezomib and ATO may be efficacious in the treatment of myeloid disorders.

Abstract 4890 Background Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells of myeloid origin, and include immature macrophages, granulocytes, dendritic cells (DC) and other myeloid cells. In mice, are phenotypically characterized as CD11b+Gr-1+. Humans MDSC have an immature phenotype, including lineage negative (Lin-), CD14-, HLA-DR-, CD15+, CD34+, CD11b+, CD33+, and CD13+ cells. MDSC impair T-cell functions through secretion immunosuppressive cytokines, perturbation of the arginine metabolism by inducible nitric oxide synthase (iNOS), up-regulation of reactive oxygen species (ROS), therefore are considered an important tumor escape mechanism. These cells, also promote tumor-dependent angiogenesis as well as tumor metastasis, and to provide tumor resistance to antiangiogenic drugs. Their accumulation has been described in the peripheral blood of patients affected by breast, lung, renal and neck carcinoma, melanoma, chronic infections, inflammatory diseases, and traumatic stress. We investigate MSDC in patients with multiple myeloma (MM) and monoclonal gammopathy undetermined significance (MGUS) by flow cytometry Methods We studied 22 patients with MM at diagnosis, 20 patients with MGUS, and 10 healthy controls (HC). Results we observed that patients with MGUS showed the same number of MDSC (CD11b+,CD13+,CD34+,CD14-,CD45+) in the peripheral blood compared to HC (1,90±1,03/mmc, p=0,23). On the contrary, patients affected by MM showed a significant increase of MDSC vs HC (6,80±8,79/mmc vs 1,45± 0,98/mmc, p=0,003). Conclusion Our results suggest that these myeloid-derived suppressor cells, through their mechanisms immunesuppressive and proangiogenesis, could be involved in the progression of MGUS towards overt MM. Disclosures No relevant conflicts of interest to declare.