The Granulocyte colony-stimulating factor produces long-term changes on gene and miRNA expression profiles in CD34+ cells from healthy donors

Granulocyte colony-stimulating factor is the most commonly used cytokine for the mobilization of hematopoietic progenitor cells from healthy donors for allogeneic stem cell transplantation. Although the administration of this cytokine is considered safe, knowledge about its long-term effects, especially in hematopoietic progenitor cells, is limited. On this background, the aim of our study was to analyze whether or not granulocyte colony-stimulating factor induces changes in gene and microRNA expression profiles in hematopoietic progenitor cells from healthy donors, and to determine whether or not these changes persist in the long-term. For this purpose, we analyzed the whole genome expression profile and the expression of 384 microRNA in CD34(+) cells isolated from peripheral blood of six healthy donors, before mobilization and at 5, 30 and 365 days after mobilization with granulocyte colony-stimulating factor. Six microRNA were differentially expressed at all time points analyzed after mobilization treatment as compared to the expression in samples obtained before exposure to the drug. In addition, 2424 genes were also differentially expressed for at least 1 year after mobilization. Of interest, 109 of these genes are targets of the differentially expressed microRNA also identified in this study. These data strongly suggest that granulocyte colony-stimulating factor modifies gene and microRNA expression profiles in hematopoietic progenitor cells from healthy donors. Remarkably, some changes are present from early time-points and persist for at least 1 year after exposure to the drug. This effect on hematopoietic progenitor cells has not been previously reported

Cannabinoid derivatives exert a potent anti-myeloma activity both in vitro and in vivo

12 p.-6 fig.-1 tab.Although hematopoietic and immune system show high levels of the cannabinoid receptor CB2, the potential effect of cannabinoids on hematologic malignancies has been poorly determined. Here we have investigated their anti-tumor effect in multiple myeloma (MM). We demonstrate that cannabinoids induce a selective apoptosis in MM cell lines and in primary plasma cells of MM patients, while sparing normal cells from healthy donors, including hematopoietic stem cells. This effect was mediated by caspase activation, mainly caspase-2, and was partially prevented by a pan-caspase inhibitor. Their pro-apoptotic effect was correlated with an increased expression of Bax and Bak, a decrease of Bcl-xL and Mcl-1, a biphasic response of Akt/PKB and an increase in the levels of ceramide in MM cells. Inhibition of ceramide synthesis partially prevented apoptosis, indicating that these sphingolipids play a key role in the pro-apoptotic effect of cannabinoids in MM cells. Remarkably, blockage of the CB2 receptor also inhibited cannabinoid-induced apoptosis. Cannabinoid derivative WIN-55 enhanced the anti-myeloma activity of dexamethasone and melphalan overcoming resistance to melphalan in vitro. Finally, administration of cannabinoid WIN-55 to plasmacytoma-bearing mice significantly suppressed tumor growth in vivo. Together, our data suggest that cannabinoids may be considered as potential therapeutic agents in the treatment of MM.Grant sponsor: Junta de Andalucía; Grant numbers: PI-0355–2013 and AC-0062–2013; Grant sponsor: Instituto de Salud Carlos III;Grant numbers: PI14/02074 and CP12/03273.Peer reviewe

Effect of pomalidomide on T cell polarization is mediated through epigenetic modifcations

Resumen del póster presentado al 22nd Congress of the European Hematology Association, celebrado en Madrid (España) del 22 al 25 de junio de 2017.-- Alvarez Laderas, Isabel et al.[Background]: There is conflicting evidence regarding the potential use of IMIDs and particularly pomalidomide after allogeneic stem cell transplantation (allo-HSCT). It has been well described that IMIDs polarize naïve T cells towards a Th1 phenotype increasing IFN-γ cytokine production via the augmentation of T-bet transcription factor. This effect might increase the risk of GvHD after allo-HSCT. Nevertheless, a recent trial has reported a potential benefit on the use of pomalidomide as GVHD treatment. [Aims]: In the current study, we have analyzed the effect of pomalidomide in the polarization of CD45RA+ cells and the epigenetic mechanisms that might be involved in this effect. [Methods]: Isolated CD45RA+ T cells from healthy donor’s Buffy Coats werestimulated with anti-CD3 plus anti-CD28 in the presence of several cytokines to polarize towards Th1 (IL-12, INF-γand anti-IL4) or Th2 (IL-4, IL-2, anti-IFN-γ and anti-IL-12) for 5 days. Pomalidomide at two different doses (10 and 100 nM) were added into the culture and the effect on T cells polarization was analyzed by flow cytometry after staining with anti-CD25, anti-IFNγ, anti-CD4 and anti-IL2 for Th1 cell polarization and anti-CD25, anti-IL10, anti-CD3 and anti-IL4 for Th2 cell polarization. In addition, the release of cytokines (IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ) in cell culture supernants were measured with BD Human Th1/Th2 Cytokine CBA kit (BDBiosciences) and T-bet and GATA-3 expression were analyzed by Western Blot. Chromatin immunoprecipitation (ChIP) assays were performed to assess the trimethylation of H3K4 (associated with gene activation) and the trimethylation of H3K27 (associated with gene repression) in the TBET and GATA-3 gene promoters. [Results]: Pomalidomide increased the expression of INF-γ and IL-2 as determined by flow cytometry in Th1 cell culture conditions. By contrast, in the presence of Th2 promoting cytokines, we observed an increase for both IL-10 and IL-4 upon adding pomalidomide to the culture. In addition, the exposure to pomalidomide increased the levels of TNF-α, INF-γ and IL-2 in the Th1 polarizing culture while, under Th2 promoting conditions, an increased concentration of IL-4 and IL-2 in supernatant was observed after exposure to pomalidomide. Furthermore, exposure to pomalidomide led to an increased expression of T-bet as assessed by western-blot in naïve CD45RA+ cells activated with anti-CD3 plus anti-CD28 and supplemented with IL-12, INF-γ and anti-IL4. By contrast, in Th2 polarization conditions, pomalidomide increased GATA-3 expression.We next studied whether or not the effect of pomalidomide in T cell polarization might be mediated by epigenetic mechanisms: in the presence of Th1 promoting conditions there was a significant increase of the activation marker H3K4me3 at the TBET promoter and a significant decrease in H3K27me3 upon exposure to the drug while, under Th2 promoting conditions, a significant increase in H3K4me3 at the promoter of GATA-3 gene was observed among T cells exposed to pomalidomide. [Conclusion]: Pomalidomide favours both Th1 and Th2 cell differentiation of CD45RA+ cells depending on the cytokines present in the medium. Treatment of naïve T cells with pomalidomide induces epigenetic modifications during T cell polarization which might favour the process of differentiation of the naïve T cells.Peer Reviewe

Efecto antileucémico de derivados cannabinoides: un nuevo grupo terapéutico en LMA

2 p.-3 fig.Los cannabinoides, componentes activos del cannabis, se emplean como tratamiento paliativo en pacientes con cáncer en virtud de su efecto antiemético y analgésico. Además, hay evidencias de que estos compuestos inhiben el crecimiento de algunas células tumorales en animales de laboratorio. Estos compuestos actúan uniéndose a receptores específicos. Los principales receptores son el CB1 (mayoritario en SNC) y CB2 (en tejido hematopoyético). El objetivo de este estudio fue confirmar el efecto terapéutico de derivados cannabinoides sintéticos específicos del receptor CB2 en LMA.Peer reviewe

Cannabinoids derivatives modify the pattern of sphingolipids in acute myeloid leukemia cells and produce a potent anti-leukemic effect

2 p. 22nd Congress of the European Hematology Association. Madrid, Spain, June 22 - 25, 2017Endocannabinoid system is a set of ligands, receptors and endogenous enzymes which modulate a variety of physiological effects. There are two well-characterized cannabinoid receptors, CB1 (mainly expressed in Central Nervous System) and CB2 (mainly in hematopoietic cells). Here, we tested the effect of the cannabinoid WIN-55 212-2 in acute myeloid leukemia (AML) in vitro and in vivo and studied the molecular signaling pathways involved in this effect, specially the role of sphingolipids. Moreover, we synthesized a new family of twelve cannabinoids that are specific to CB2 receptor.Peer reviewe

The Granulocyte colony-stimulating factor produces long-term changes in gene and microRNA expression profiles in CD34+ cells from healthy donors

Granulocyte colony-stimulating factor is the most commonly used cytokine for the mobilization of hematopoietic progenitor cells from healthy donors for allogeneic stem cell transplantation. Although the administration of this cytokine is considered safe, knowledge about its long-term effects, especially in hematopoietic progenitor cells, is limited. On this background, the aim of our study was to analyze whether or not granulocyte colony-stimulating factor induces changes in gene and microRNA expression profiles in hematopoietic progenitor cells from healthy donors, and to determine whether or not these changes persist in the long-term. For this purpose, we analyzed the whole genome expression profile and the expression of 384 microRNA in CD34(+) cells isolated from peripheral blood of six healthy donors, before mobilization and at 5, 30 and 365 days after mobilization with granulocyte colony-stimulating factor. Six microRNA were differentially expressed at all time points analyzed after mobilization treatment as compared to the expression in samples obtained before exposure to the drug. In addition, 2424 genes were also differentially expressed for at least 1 year after mobilization. Of interest, 109 of these genes are targets of the differentially expressed microRNA also identified in this study. These data strongly suggest that granulocyte colony-stimulating factor modifies gene and microRNA expression profiles in hematopoietic progenitor cells from healthy donors. Remarkably, some changes are present from early time-points and persist for at least 1 year after exposure to the drug. This effect on hematopoietic progenitor cells has not been previously reported

Polyphenolic Extract (PE) from Olive Oil Exerts a Potent Immunomodulatory Effect and Prevents Graft-versus-Host Disease in a Mouse Model

9 FigurasPolyphenols are a group of chemical substances found in plants, with immunomodulatory, antiproliferative, and anti-inflammatory properties that might be useful in the prophylaxis and treatment of graft-versus-host disease (GVHD). Polyphenolic extract (PE) obtained from extra virgin olive oil (EVOO) decreased the activation and proliferation of activated T cells. In addition, a decreased production of proinflammatory cytokines was observed upon exposure to PE. Western blot assays showed a marked inhibition of Akt phosphorylation and nuclear translocation of NF-κB in activated T cells. In a murine model of acute GVHD, we observed that mice that received a diet supplemented in PE (600 ppm) presented a higher survival rate and lower risk of developing GVHD when compared with the group that received a control diet. Histopathologic examination showed a significantly lower gut involvement in mice receiving PE, with a decrease in proinflammatory cytokines (IL-2, IL-17, and TNF-α) in serum and the reestablishment of butyrate concentration in the gut. In conclusion, PE obtained from EVOO exerted a potent immunomodulatory effect, reducing the activation and proliferation of activated T cells and the production of proinflammatory cytokines. In a murine model of acute GVHD, a PE-supplemented diet reduced the incidence and severity of the disease and increased survival after transplantation.Peer reviewe

Cannabinoid derivatives exert a potent anti-myeloma activity both in vitro and in vivo.

Although hematopoietic and immune system show high levels of the cannabinoid receptor CB2, the potential effect of cannabinoids on hematologic malignancies has been poorly determined. Here we have investigated their anti-tumor effect in multiple myeloma (MM). We demonstrate that cannabinoids induce a selective apoptosis in MM cell lines and in primary plasma cells of MM patients, while sparing normal cells from healthy donors, including hematopoietic stem cells. This effect was mediated by caspase activation, mainly caspase-2, and was partially prevented by a pan-caspase inhibitor. Their pro-apoptotic effect was correlated with an increased expression of Bax and Bak, a decrease of Bcl-xL and Mcl-1, a biphasic response of Akt/PKB and an increase in the levels of ceramide in MM cells. Inhibition of ceramide synthesis partially prevented apoptosis, indicating that these sphingolipids play a key role in the pro-apoptotic effect of cannabinoids in MM cells. Remarkably, blockage of the CB2 receptor also inhibited cannabinoid-induced apoptosis. Cannabinoid derivative WIN-55 enhanced the anti-myeloma activity of dexamethasone and melphalan overcoming resistance to melphalan in vitro. Finally, administration of cannabinoid WIN-55 to plasmacytoma-bearing mice significantly suppressed tumor growth in vivo. Together, our data suggest that cannabinoids may be considered as potential therapeutic agents in the treatment of MM