ALBERT

Description: Mesenchymal stromal cells (MSCs), an essential element of both normal and leukemic hematopoietic microenvironment, are multipotent cells with a unique immune-modulating ability. Thus, MSCs play a crucial role for both the proliferation and differentiation of hematopoietic stem cells (HSCs) and induce an immune-tolerant milieu. Indoleamine 2, 3-dioxygenase (IDO1 and IDO2) enzymes catabolize tryptophan to kynurenines and play a key role in the induction of immune tolerance in different settings, including acute myeloid leukemia (AML). Furthermore, IDO1/IDO2 pathway is a well described mechanism by which MSCs exert their mmunomodulatory properties. We hypothesized that: 1) MSC-dependent mechanisms are involved in leukemia initiation, maintenance and progression; 2) the expression of IDO1 and IDO2 by MSCs is part of a MSC-dependent mechanism able to create a tumor-supportive milieu. To this aim, we isolated MSCs from the bone marrow of AML patients (AML-MSCs) at diagnosis. We first analyzed their phenotypic and functional properties compared to that of healthy donor-derived MSCs (HD-MSCs). We found that AML-MSCs showed a reduced proliferative capacity but normal immunophenotype, differentiative and immunomodulatory capacity as compared to HD-MSCs. Furthermore, AML-MSCs did not show the chromosomal abnormalities identified in the primary blast counterpart (FISH analysis). We next investigated IDO1/2 expression and functions in MSCs. We demonstrated that IDO enzymes are expressed in AML-MSCs as well as in HD-MSCs. IDO1 is efficiently upregulated by different inflammatory stimuli, and IDO1 protein expression parallels mRNA in both HD-MSCs and AML-MSCs. Interestingly, IDO2 mRNA is expressed at low basal level in all analyzed conditions in HD-MSCs, while it is upregulated, in particular after IFN-gamma stimulation, in AML-MSCs, although the level of induction varies between different patients. When T-cell proliferation was tested in MSC co-cultures, w/or w/out IDO1/2 inhibitor, 1-methyltryptophan, we found that MSC immunomodulatory potential is IDO-dependent both in HD-MSCs and AML-MSCs. Finally, we found that in co-culture assay with primary AML blasts, MSCs stimulated blast proliferation and this effect is, at least in part, IDO-mediated. These data suggested that IDO enzymes, in particular IDO2, may be differentially expressed in AML-MSCs as compared to HD-MSCs and IDO inhibition has an impact on MSC/AML cell cross talk. These findings may help to discover novel niche-target prognostic/therapeutic factors and to provide novel applications for drugs already under active clinical investigation (i.e. IDO-inhibitors). Disclosures Cavo: Janssen-Cilag, Celgene, Amgen, BMS: Honoraria.

Description: Abstract 1047 Indoleamine 2,3-dioxygenase (IDO) enzymes (IDO1 and IDO2) critically regulate the rate-limiting step of tryptophan degradation along kynurenine pathway in several cellular subsets, including dendritic cells (DCs). In particular, it is known that IDO1 expression in DCs is induced by inflammatory mediators, especially PGE2, and enhances DCs tolerogenic capacity, such as the induction of T regulatory cells (Tregs). In absence of exogenous stimuli, IDO1 is driven to proteasomal degradation by SOCS3. Conversely, few and contrasting results are available about IDO2 expression and function in human DCs. We, then, besides IDO1, investigated the expression of IDO2 in human DC subsets, obtained from blood samples of healthy volunteers. Circulating myeloid and plasmacytoid DCs were purified and analyzed for the expression of both IDO enzymes. Our data demonstrate that myeloid DCs express higher levels of both IDO1 and IDO2 mRNA in comparison to plasmacytoid DCs. Moreover, PGE2 modulates IDO1 and IDO2 mRNA expression only in myeloid DCs and has no effect in plasmacytoid DCs. At protein level, IDO1 is expressed only in myeloid DCs and is modulated by PGE2, whereas IDO2 is expressed in both myeloid and plasmacytoid DCs and is not modulated by PGE2. Interestingly, when gene transcription or protein translation are inhibited, IDO2 protein expression persists independently from PGE2, while IDO1 expression requires PGE2 presence and needs continuous transcription and translation. Such discrepancy might be derived by a different binding of IDO1 and IDO2 to SOCS3, which in absence of pro-inflammatory stimuli is known to bind IDO1 and to drive it to proteasomal degradation. Indeed, SOCS3 immunoprecipitation and proteasomal inhibition experiments show that SOCS3 does not bind IDO2 which is not, therefore, driven to proteasomal degradation and is stably expressed. At the functional level, both myeloid and plasmacytoid DCs generate Tregs through an IDO-dependent mechanism, since the addition of IDO inhibitors 1-MT-D and 1-MT-L abrogate Tregs generation. More specifically, the silencing of IDO2 through its specific siRNA downregulates kynurenine production and Tregs generation, thus demonstrating that IDO2 is functionally active in DCs. In conclusion, our data demonstrate that, besides IDO1, human DCs also express IDO2, which contributes to tolerance by degrading tryptophan into kynurenine and by inducing Tregs. However, unlike IDO1, IDO2 expression is independent from inflammatory mediators, such as PGE2, and does not require continuous synthesis. These findings indicate IDO pathway as a critical check-point in the fine-tuning of DC-mediated tolerance induction. In particular, while IDO2 expression may confer a homeostatic tolerogenic capacity to DCs upon steady-state conditions, inflammation results in the induction of additional mechanisms of immune tolerance, such as the up-regulation of IDO1. Disclosures: Metz: New Link Genetics Corporation: Employment.

Description: Abstract 3231 The indoleamine 2,3-dioxygenases IDO1 and IDO2 participate in tryptophan catabolism along the kynurenine pathway and are specifically inhibited by 1-methyltryptophan (1-MT). IDO1 expression in dendritic cells (DCs) is upregulated by various maturation stimuli. In particular, PGE2 plays a crucial role in inducing IDO1-expressing DCs. While it is well established that IDO1-expressing DCs contribute to immunological tolerance by a number of mechanisms, little is known about the expression, function and regulation of IDO2 in DCs. Here we show that immature monocyte-derived DCs (Mo-DCs) express IDO2 which, like IDO1, is also upregulated upon maturation with a prostaglandin-E2 (PGE2)-containing cytokine cocktail. However, while IDO1 upregulation is strictly dependent on PGE2 during maturation, IDO2 expression level is not affected by PGE2. We show that IDO2 expressed by Mo-DCs is enzymatically active, producing L-kynurenine, and that this activity affects T-cell functions by inhibiting allogeneic and autologous T-cell proliferation and by inducing formation of a population of T regulatory cells (Tregs). Taken together, our findings establish different regulatory pathways for IDO2 and IDO1 in Mo-DCs, where IDO2 is functionally active, and they demonstrate that the tolerogenic function of Mo-DCs relies in part on PGE2-independent expression of IDO2. Disclosures: Metz: New Link Genetics Corporation: Employment. Prendergast:New Link Genetics Corporation: Employment.

Description: Abstract 3848 Introduction: Human bone marrow derived Mesenchymal Stem Cells (hMSCs) are adult multipotent cells. hMSCs differentiate in vitro and in vivo into several tissue lineages originating from the three germinal layers making them attractive candidates for bioengineering and cellular therapy. Thus, it seems of great relevance to search putative messengers and signalling able to modulate their proliferation and differentiation. Nucleotides triphosphates are extracellular messengers binding to specific receptors (P2Rs) that modulate cell functions depending on the cell type. Controversial information is available on P2 expression and activity in hMSCs. Methods and Results: Here we found that hMSCs expressed several P2R subtypes. hMSCs were very resistant to the cytotoxic effects of high concentrations of ATP, as demonstrated by the lack of morphological and mitochondrial changes or release of intracellular markers of cell death. Gene expression profiling revealed that ATP treatment down-regulated cell proliferation and up-regulated cell migration genes in hMSCs. Functional studies confirmed the inhibitory activity of ATP on proliferation and clonogenic ability of hMSCs. Furthermore, ATP potentiated the chemotactic response of hMSCs to the chemokine CXCL12, and increased their spontaneous migration. In vivo, xenotransplant experiments showed that the homing capacity of hMSCs to murine bone marrow was increased by ATP pre-treatment. Moreover, ATP increased pro-inflammatory cytokines production (IL-2, IFN-g, IL-12p70), while decreased secretion of the anti-inflammatory cytokine IL-10. This finding was associated with the reduced ability of ATP-treated hMSC of inhibiting T-cell proliferation. Microarrays data suggested that several genes implicated in hMSC differentiation can be modulated by ATP treatment. To further investigate this issue, hMSCs cells were cultured under adipogenic or osteogenic conditions and were transiently exposed to ATP before starting differentiation or continuously exposed to ATP for the first 3 days of differentiation induction. We demonstrated that adipogenesis-related accumulation of lipids, analyzed by Oil red O staining, was more evident in ATP treated cultures. Furthermore, quantitative real time PCR (qRT-PCR) assay showed that mRNA expression of PPARg, a transcription factor early up-regulated during adipogenesis, was significantly increased in hMSCs differentiated cells treated with ATP. In osteogenic condition, analysis of mineralized area through Alizarin Red staining, indicated that ATP treatment enhanced the extent of mineralization compared to untreated control. The expression of RUNX2, a key transcription factor in osteogenesis, analyzed by qRT-PCR in differentiated cells confirmed data obtained in Alizarin-based assay. Conclusions: These data demonstrated that purinergic signalling modulates biological functions and differentiation potential of hMSCs. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction. Mesenchymal stromal cells (MSCs) substantially contribute to the creation of hematopoietic niche by regulating hematopoietic stem cell (HSC) fate and have a unique immune-modulating capacity. In the leukemic milieu, the presence of MSCs constitutes a side effect, since MSCs not only favor leukemic cell survival, but they can also generate an immune-tolerant environment. Although recent findings have outlined a putative MSC role in hematological malignancy development, MSC-dependent mechanisms potentially supporting leukemia remain unclear. We hypothesize that leukemic cells can shape bone marrow (BM) MSCs by inducing functional changes, able to convert the BM microenvironment from hostile to permissive for leukemia. Methods. We isolated acute myeloid leukemia (AML) cells and generated AML-MSCs from the BM of AML patients. Next, we set up AML-MSC/AML cell co-culture experiments and we investigated gene expression in AML-MSCs and AML cells before and after co-cultures. Results. Our microarray data on BM isolated cells (AML patients, N=61; healthy donors, N=7) indicated Interferon(IFN)-γ as un up-regulated gene in almost 40% of AML samples. Furthermore, multivariate analysis, showed that IFN-g-positive AML patients had a better overall survival. Thus, we decided to deepen IFN-ϒ-dependent modifications in leukemic milieu through in vitro studies. In AML-MSC/AML cell co-culture experiments, we confirmed microarray data and we found that AML cells produced IFN-γ. We next demonstrated that indoleamine 2,3-dioxygenase (IDO)1 enzyme, a master regulator of MSC immune suppressive functions, is up-regulated in AML-MSCs after co-culture with IFN-γ-producing AML cells. Such effect was abrogated by adding to cell cultures an IFN-γ neutralizing antibody. Finally, we found that AML-MSCs, after co-culture with IFN-γ-producing AML cells, were able to induce regulatory T cell in a IDO1-dependent manner. To gain further insight in AML cell-dependent MSC modifications, we analyzed MSC expression of IFN-γ-stimulated genes (ISGs) such as Programmed death-ligand (PDL)-1 and Nitric Oxide synthase (NOS)-2 which are known to regulate immunity and promote tolerance. In particular, we tested the ISG expression in MSCs after co-cultures with IFN-ϒ positive or IFN-γ negative AML cells. We found that IFN-γ positive, but not IFN-γ negative AML cells, were able to induce PDL-1 and NOS2 in AML-MSCs. Thus, ISG expression profile in AML-MSCs after co-cultures with IFN-γ positive AML cells was similar as that observed in MSCs after exposure to recombinant IFN-γ. Conclusions. Our data suggest that inflammatory signals produced by AML cells are able to modify MSC functions, thus favoring an immune-tolerant and leukemia supporting milieu. Overall, our results would likely contribute to unravel MSC-dependent mechanisms promoting leukemia and will help to provide novel applications for drugs already under experimentation (e.g. IDO-inhibitors, Checkpoint inhibitors) to translate into more effective therapies in AML patients. Disclosures Cavo: AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Description: Introduction: ELN intermediate-risk AML poses considerable challenges to clinicians both in terms of accurate prognostication and optimal treatment. Indoleamine 2,3-dioxygenase 1 (IDO1) plays a central role as a mediator of immune tolerance in AML through the increase of Treg cells. IDO1 activity is negatively regulated by the BIN1 proto-oncogene. Herein, we analyzed the correlation between BIN1 and IDO1 expression in AML, also focusing on IDO1-interacting genes, with the aim to identify a predictive gene signature for OS. Methods: Biological and clinical data of 732 patients with de novo AML were retrieved from public TCGA and HOVON datasets. Since details on chemotherapy regimens were not available in the HOVON dataset, we decided to exclude patients 〉= 65 years from survival analyses. IDO1-interacting genes were selected through a co-expression analysis performed on TCGA RNA-sequencing data accessed through cBioPortal. The best genes combination predicting overall survival was plotted in a gene expression score. Patients were split in three different groups using score quartiles as cut-off. Results: In the HOVON dataset, IDO1 and BIN1 mRNA expression were negatively correlated (r = -0.40, P

Description: Background Acute myeloid leukemia (AML) has been considered for a long time exclusively driven by critical mutations in hematopoietic stem cells (HSCs). Recently, the contribution of bone marrow (BM) microenvironment has gained increasing attention, challenging the evidence that AML derives exclusively from leukemic cell-intrinsic defects. Mesenchymal stromal cells (MSCs) are a key component of the BM microenvironment by regulating HSC fate and having a unique immune-modulatory capacity mostly mediated by interferon (IFN)-γ-induced indoleamine 2,3-dioxygenase (IDO)-1 enzyme activity. New studies demonstrated that the alterations of MSCs are able (e.g. by promoting an inflammatory/genotoxic microenvironment) to induce hematological diseases in mice models and humans. Moreover, AML cells seem to exploit MSC-dependent pro-survival signals to their advantage. All these concepts converge to indicate a fundamental bi-directional interaction among malignant cells and BM microenvironment contributing to AML onset and progression. The mechanisms underlying this crosstalk have just been started to get unraveled. Among signals potentially driving the remodeling of the BM microenvironment, inflammation, a hallmark of cancer, seems to play a role. We hypothesize that 'inflammatory features' of leukemic cells can shape MSCs by inducing functional changes able to create a permissive/self-reinforcing niche favorable to escape therapy and immune response. Methods We isolated acute myeloid leukemia (AML) cells and generated AML-MSCs from the BM of AML patients. Gene expression profile (GEP) (AML, N=61; healthy donors, HDs N=7) and NanoString analysis (AML, N=24) on BM-derived cells were also done. Next, we set up AML-MSC/AML cell co-culture experiments and we investigated gene expression in AML-MSCs and AML cells before and after co-cultures. We also set up a murine model in which the IFN-γ expressing C1498 AML cells was knock down (KD) for the IFN-γ gene by RNA interference. BM infiltrate was analyzed in mice and AML patients. Results In a GEP-screening, we found that almost 40% of AML samples showed an IFN-γ expression higher than the median level of IFN-γ expression in HDs. NanoString data and pathway analysis indicated that IFN-γ high AML cells (above the median level) presented an inflammatory/immune modulating signature clearly distinct from IFN-γ low AML cells (below the median level). Moreover, IFN-γ expression in AML samples correlated with the up-regulation of IFN-γ-stimulated genes (ISGs) (e.g. IDO-1, Programmed death-ligand (PDL)-1 and Nitric Oxide synthase (NOS)-2), which are known to regulate immunity and tolerance. Thus, we aimed to gain insights into IFN-γ-dependent modifications in the leukemic milieu. In AML-MSC/AML cell co-culture experiments, we detected that AML cells produced IFN-γ. To gain insight in AML cell-dependent MSC modifications, we analyzed ISG expression in MSCs, after co-cultures with IFN-γ high or IFN-γ low AML cells. We found that IFN-γ high, but not IFN-γ low AML cells, were able to induce IDO-1, PDL-1 and NOS-2 in AML-MSCs. Moreover, ISG upregulation was abrogated by an IFN-γ neutralizing antibody. We also found that AML-MSCs, after co-culture with IFN-γ high AML cells, were able to induce regulatory T cells (Tregs) in an IDO1-dependent manner. In vivo experiments showed a higher percentage of engraftment in immunocompetent mice injected with parental IFN-γ expressing cells compared to mouse injected with the KD counterpart. The take of parental C1498 cells was associated to an increased frequency of Tregs in the BM. Furthermore, the BM microenvironment of mice injected with IFN-γ KD-C1498 cells showed a significant reduction of PD-L1 expressing cells. Consistently, BM infiltrate analysis in AML patients showed that the percentage of Tregs was correlated with the percentage of AML IFN-γ-positive cells in the BM. Conclusion Our data suggest that interferon-γ-dependent inflammatory signals produced by AML cells are able to modify MSC functions, thus favoring an immune modulating and leukemia-supporting milieu. Overall, our results unravel MSC-dependent mechanisms that might promote leukemia resistance to therapy, therefore informing the delivery of novel therapies targeting the AML microenvironment such as IDO inhibitors and immune checkpoint blockade. Disclosures Martinelli: Novartis: Consultancy, Other: trial grant; Daiichi Sankyo: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Other: trial grant; Incyte: Consultancy, Other: trial grant; Janssen: Consultancy, Other: trial grant; Roche: Consultancy, Other: trial grant; Amgen: Consultancy, Other: trial grant; Ariad: Consultancy, Other: trial grant; Celgene: Consultancy, Honoraria, Other: trial grant; Pfizer: Consultancy, Other: trial grant. Rutella:Kura Oncology: Research Funding; NanoString Technologies, Inc.: Research Funding; MacroGenics, Inc.: Research Funding. Cavo:bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; novartis: Honoraria.

Description: Acute Myeloid Leukemia (AML) is a clonal disease sprouting from a rare population of leukemic stem cells. Over the past years, increasing interest is gaining the contribution that cell-extrinsic factors have in AML generation and maintenance. In this context, the ability of leukemia cells to detect changes in the microenvironment is important in responsiveness to environmental fluctuations. Bitter taste receptors (T2Rs) are typical G-protein coupled receptors and are normally found on the surface of the tongue. Recent studies showed that T2Rs are widely expressed in various parts of human anatomy and have been shown to be involved in physiology of respiratory system, gastrointestinal tract and endocrine system. thus suggesting a wider function in "sensing microenvironment". We recently reported that AML cell lines OCI-AML3, THP-1, and AML primary cells expressed fully functional T2Rs subtypes. Gene expression profile analysis showed that after T2Rs activation, leukemic cell lines underwent down-regulation of genes involved in positive regulation of cell proliferation, migration, and cell-cycle. Whereas genes involved in cell adhesion and DNA repair were up-regulated. Functional assays supported these results (Blood 2017 130:3949). In the present work, we further investigated the role of T2Rs in BM microenvironment by extending the analysis to AML primary samples and to normal hematopoietic stem cells (HSCs). Similarly to AML cell lines, T2Rs activation with high dose of agonist induced a reduction of cell viability associated to apoptosis induction, while non-toxic doses reduced cell migration and clonogenic capacity. In addition, T2Rs stimulation with agonist makes AML cell lines more prone to oxidative and metabolic stress. Leukemia cells displayed a quiescent phenotype in response to T2Rs activation suggesting that mitochondrial activity is significantly limited by T2Rs agonist treatment. Since no data are available on the presence and the function of T2Rs on normal hematopoietic stem cell counterpart, we characterized T2Rs expression on CD34+ cell isolated from healthy donor. CD34+ cells express several T2Rs subtype without significant differences compare to AML cells. Their activation with high dose of agonist reduced HSCs viability inducing apoptosis, while non-cytotoxic doses reduced clonogenic capacity and promoted migration. Given the effect of T2Rs activation on crucial AML cell function, we tested the therapeutical potential of T2R agonist with and without conventional chemioterapic agent. Interestingly we observed that T2Rs agonist have a synergistic effect with cytarabine, reducing leukemia cell viability when combined with ARA-C compared to their use as single compound. The combination allowed to reach a high toxicity using lower doses of chemotherapic agent. Overall our results indicate that T2Rs receptor system is expressed and functional in both leukemic cells and HSCs. In particular, in AML cells T2Rs activation is associated with quiescence induction and prevention of migration. T2Rs stimulation modulates HSCs function but their role need to be further deepen. These data may suggest a role for microenvironment "bitter" molecules in regulating normal and leukemic hematopoiesis. Disclosures Cavo: AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.