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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.

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.