MacroH2A1.1 as a crossroad between epigenetics, inflammation and metabolism of mesenchymal stromal cells in myelodysplastic syndromes.

Ineffective hematopoiesis is a hallmark of myelodysplastic syndromes (MDS). Hematopoietic alterations in MDS patients strictly correlate with microenvironment dysfunctions, eventually affecting also the mesenchymal stromal cell (MSC) compartment. Stromal cells are indeed epigenetically reprogrammed to cooperate with leukemic cells and propagate the disease as "tumor unit"; therefore, changes in MSC epigenetic profile might contribute to the hematopoietic perturbations typical of MDS. Here, we unveil that the histone variant macroH2A1 (mH2A1) regulates the crosstalk between epigenetics and inflammation in MDS-MSCs, potentially affecting their hematopoietic support ability. We show that the mH2A1 splicing isoform mH2A1.1 accumulates in MDS-MSCs, correlating with the expression of the Toll-like receptor 4 (TLR4), an important pro-tumor activator of MSC phenotype associated to a pro-inflammatory behavior. MH2A1.1-TLR4 axis was further investigated in HS-5 stromal cells after ectopic mH2A1.1 overexpression (mH2A1.1-OE). Proteomic data confirmed the activation of a pro-inflammatory signature associated to TLR4 and nuclear factor kappa B (NFkB) activation. Moreover, mH2A1.1-OE proteomic profile identified several upregulated proteins associated to DNA and histones hypermethylation, including S-adenosylhomocysteine hydrolase, a strong inhibitor of DNA methyltransferase and of the methyl donor S-adenosyl-methionine (SAM). HPLC analysis confirmed higher SAM/SAH ratio along with a metabolic reprogramming. Interestingly, an increased LDHA nuclear localization was detected both in mH2A1.1-OE cells and MDS-MSCs, probably depending on MSC inflammatory phenotype. Finally, coculturing healthy mH2A1.1-OE MSCs with CD34+ cells, we found a significant reduction in the number of CD34+ cells, which was reflected in a decreased number of colony forming units (CFU-Cs). These results suggest a key role of mH2A1.1 in driving the crosstalk between epigenetic signaling, inflammation, and cell metabolism networks in MDS-MSCs.

High-density neutrophils in MGUS and multiple myeloma are dysfunctional and immune-suppressive due to increased STAT3 downstream signaling.

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.

Biological properties of Cakile maritima Scop. (Brassicaceae) extracts.

OBJECTIVE: Cakile maritima scop. (CKM) is a herbaceous plant (Brassicaceae) growing also in high salinity environment. It is an annual plant growing in clumps or mounds in the sand on beaches and bluffs. MATERIALS AND METHODS: Stems, seeds, leaves and flowers of CKM were used to obtain 70% of ethanol extracts. The phenolic content of the different extracts was evaluated by the Folin-Ciocalteu method. The separation of phytochemical compounds was based on ultra-performance liquid chromatography coupled to mass spectrometry. Radical scavenging activity was determined by 1,1-diphenyl-2-picrylhydrazyl assay. The qualitative assay for the inhibition of α-glucosidase was quantified spectrophotometrically and the anti-inflammatory activity was determined in the U937 cell line by using gene expression of pro-inflammatory cytokines. Cell viability assay was done in U937, MM1S, and U266 cells by using the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. The antimicrobial activity was investigated by MIC determination, "double-triple combinations assay", and growth inhibition curves analysis, using the extracts individually or in various combination. Statistical analysis was performed by the Student's t-test and ANOVA. RESULTS: All parts of the plant exhibited a high antioxidant capacity as measured by DPPH assay. Furthermore, all extracts reduced (about 10 folds) the expression of inflammatory cytokines in macrophage following LPS treatment. As regards the antibacterial activity, only the seeds extract was able to inhibit both Gram-negative and Gram-positive bacteria when tested alone, whereas dual combinations of different extracts (leaves, flowers, stems and seeds) caused bacterial inhibition exhibiting a synergic action. Finally, we showed that the extracts did not exhibit cytotoxic effects in normal cells and that, surprisingly, it exhibited an anti-proliferative effect (inhibition ≈80%) in multiple myeloma U266 cells. CONCLUSIONS: Our study suggests that CKM possesses antioxidant, anti-inflammatory, antibacterial, anti-proliferative activities and such pleiotropic effects may be exploited under various pathological conditions.

BRIT1/MCPH1 expression in chronic myeloid leukemia and its regulation of the G2/M checkpoint.

BRIT1 (BRCT-repeat inhibitor of hTERT expression), also known as microcephalin (MCPH1), is a crucial gene in the complex cellular machine that is devoted to DNA repair and acts as a regulator of both the intra-S and G2/M checkpoints. The most important role of BRIT1/MCPH1 in the regulation of cell cycle progression appears to be the G2/M checkpoint. The K562 and peripheral blood cells of chronic myeloid leukemia (CML) patients at diagnosis were found to downregulate BRIT1/MCPH1. However, we could not find any correlation between bcr/abl activity and the BRIT1/MCPH1 level. In order to study the genomic instability of CML cells, we evaluated the ability of these cells to arrest mitotic division after exposure to hydroxyurea, a known genotoxic agent. We showed that CML cells continue to proliferate without the activation of the G2/M cell cycle checkpoint arrest or of the apoptotic mechanism. This behavior may predispose the cells to accumulate genomic defects. In conclusion, we found that CML cells have a low BRIT1/MCPH1 level and show a defective G2/M arrest, confirming that these cells have a constitutive genomic instability.