ALBERT

Description: The Journal of Organic Chemistry DOI: 10.1021/acs.joc.8b00488

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: Growing evidence highlights the endocannabinoid (EC) system involvement in cancer progression. Lipid mediators of this system are secreted by hematopoietic cells, including the ECs 2-arachidonoyl-glycerol (2AG) and arachidonoyl-ethanolamide (AEA), the 2AG metabolite 1AG, and members of N-acylethanolamine (NAE) family—palmitoyl-ethanolamide (PEA) and oleoyl-ethanolamide (OEA). However, the relevance of the EC system in myeloproliferative neoplasms (MPN) was never investigated. We explored the EC plasma profile in 55 MPN patients, including myelofibrosis (MF; n = 41), polycythemia vera (PV; n = 9), and essential thrombocythemia (ET; n = 5) subclasses and in 10 healthy controls (HC). AEA, PEA, OEA, 2AG, and 1AG plasma levels were measured by LC–MS/MS. Overall considered, MPN patients displayed similar EC and NAE levels compared to HC. Nonetheless, AEA levels in MPN were directly associated with the platelet count. MF patients showed higher levels of the sum of 2AG and 1AG compared to ET and PV patients, higher OEA/AEA ratios compared to HC and ET patients, and higher OEA/PEA ratios compared to HC. Furthermore, the sum of 2AG and 1AG positively correlated with JAK2V617F variant allele frequency and splenomegaly in MF and was elevated in high-risk PV patients compared to in low-risk PV patients. In conclusion, our work revealed specific alterations of ECs and NAE plasma profile in MPN subclasses and potentially relevant associations with disease severity.

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: Introduction A large body of evidence has increasingly demonstrated that the immune tumor microenvironment (TME) critically contributes to acute myeloid leukemia (AML) development. However, current AML prognostic classifications only rely on leukemia cell-intrinsic alterations and do not incorporate immunological markers referring to TME. Indoleamine 2,3-dioxygenase 1 (IDO1), which is negatively regulated by the BIN1 proto-oncogene, is a central mediator of immune tolerance in the AML TME. This study aimed to identify IDO1-interacting genes in the AML TME and to develop a prognostic immune gene signature. Methods Biological and clinical data of 732 patients with de novo AML treated with curative intent were retrieved from public TCGA and HOVON datasets. Patients 〉= 65 years were excluded from survival analyses. Co-expression analysis was performed through cBioPortal on TCGA data aiming at discovering new IDO1-interacting genes. Cox regression analysis was used to identify most survival-predicting genes in order to generate a prognostic score. Differential expression (DE) analysis was performed using the nSolver software package (NanoString Technologies, Seattle, WA). Results BIN1 and IDO1 expression were negatively correlated in HOVON cases (P