Although the role of CD4+ T cells and in particular Tregs and Th17 cells is established in myelodysplastic syndrome(MDS), the contribution of other components of immune system is yet to be elucidated fully. In this study we investigated the number and function of myeloid derived suppressor cells (MDSCs) in fresh peripheral blood and matched bone marrow samples from 42 MDS patients and the potential correlation with risk of disease progression to acute myeloid leukemia (AML). In peripheral blood, very low-/low risk patients had significantly lower median MDSC number (0.16×109/L(0.03–0.40)) compared to intermediate-/high-/very high risk patients, in whom median MDSC counts was 0.52×109/L(0.10–1.78), p < 0.005. When co-cultured with CD4+ effector T-cells (T-effectors), MDSCs suppress Teffector proliferation in both allogeneic and autologous settings. There was a positive correlation between the number of Tregs and MDSCs (Spearman R = 0.825, p < 0.005) in high risk and not low risk patients. We also investigated MDSCs' expression of bone marrow-homing chemokine receptors, and our data shows that MDSCs from MDS patients express both CXCR4 and CX3CR1 which might facilitate migration of MDSCs to bone marrow. Monocytic MDSCs(M-MDSCs) which are more frequent in the peripheral blood express higher levels of CX3CR1 and CXCR4 than the granulocytic subtype (G-MDSCs), and circulating M-MDSCs had significantly higher CX3CR1 expression compared to bone-marrow M-MDSCs in intermediate-/high-/very high risk MDS. Our results suggest that MDSCs contribute significantly to the dysregulation of immune surveillance in MDS, which is different between low and high risk disease. It further points at mechanisms of MDSCs recruitment and contribution to the bone marrow microenvironment.

Interactions between acute myeloid leukemia (AML) blasts and neighboring stromal cells are important for disease development and chemosensitivity. However, the molecular mechanisms involved in the cytokine-mediated crosstalk between mesenchymal stem cells (MSCs) and AML cells are largely unknown. Leukemic cells derived from 18 unselected AML patients were cultured with bone marrow MSCs derived from healthy donors; the populations then being separated by a semipermeable membrane. Coculture had only minor effects on MSC proliferation. The unique cytokine network in cocultures was determined by high constitutive MSC release of certain cytokines (especially IL-6 and vascular endothelial growth factor) and constitutive release of a wide range of soluble mediators by primary AML cells. However, the AML cell release varied considerably between patients, and these differences between patients were also reflected in the coculture levels even though supra-additive effects were seen for many mediators. These effects on the local cytokine network were dependent on a functional crosstalk between the two cell subsets. The crosstalk altered the global gene expression profile of the MSCs, especially expression of genes encoding proteins involved in downstream signaling from Toll like receptors, NFκB signaling and CCL/CXCL chemokine release. Thus, primary AML cells alter the functional phenotype of normal MSCs.

The phosphatidylinositol 3-kinase (PI3K)-Akt-mechanistic target of rapamycin (mTOR) pathway is constitutively activated in human acute myeloid leukemia (AML) cells and is regarded as a possible therapeutic target. Insulin is an agonist of this pathway and a growth factor for AML cells. We characterized the effect of insulin on the phosphorylation of 10 mediators in the main track of the PI3K-Akt-mTOR pathway in AML cells from 76 consecutive patients. The overall results showed that insulin significantly increased the phosphorylation of all investigated mediators. However, insulin effects on the pathway activation profile varied among patients, and increased phosphorylation in all mediators was observed only in a minority of patients; in other patients, insulin had divergent effects. Global gene expression profiling and proteomic/phosphoproteomic comparisons suggested that AML cells from these two patient subsets differed with regard to AML cell differentiation, transcriptional regulation, RNA metabolism, and cellular metabolism. Strong insulin-induced phosphorylation was associated with weakened antiproliferative effects of metabolic inhibitors. PI3K, Akt, and mTOR inhibitors also caused divergent effects on the overall pathway phosphorylation profile in the presence of insulin, although PI3K and Akt inhibition caused a general reduction in Akt pT308 and 4EBP1 pT36/pT45 phosphorylation. For Akt inhibition, the phosphorylation of upstream mediators was generally increased or unaltered. In contrast, mTOR inhibition reduced mTOR pS2448 and S6 pS244 phosphorylation but increased Akt pT308 phosphorylation. In conclusion, the effects of both insulin and PI3K-Akt-mTOR inhibitors differ between AML patient subsets, and differences in insulin responsiveness are associated with differential susceptibility to metabolic targeting.

Subclassification of AML patients based on the constitutive cytokine release may be clinically relevant and a part of a low-risk (i.e. chemosensitive) AML cell phenotype.

ABSTRACT:The retinoid-responsive gene CXXC5 localizes to the 5q31.2 chromosomal region and encodes a retinoid-inducible nuclear factor (RINF) that seems important during normal myelopoiesis. We investigated CXXC5/RINF expression in primary human acute myeloid leukemia (AML) cells derived from 594 patients, and a wide variation in CXXC5/RINF mRNA levels was observed both in the immature leukemic myeloblasts and in immature acute lymphoblastic leukemia cells. Furthermore, patients with lowrisk cytogenetic abnormalities showed significantly lower levels compared to patients with high-risk abnormalities, and high RINF/CXXC5/ mRNA levels were associated with decreased overall survival for patients receiving intensive chemotherapy for newly diagnosed AML. This association with prognosis was seen both when investigating (i) an unselected patient population as well as for patients with (ii) normal cytogenetic and (iii) core-binding factor AML. CXXC5/RINF knockdown in AML cell lines caused increased susceptibility to chemotherapy-induced apoptosis, and regulation of apoptosis also seemed to differ between primary human AML cells with high and low RINF expression. The association with adverse prognosis together with the antiapoptotic effect of CXXC5/RINF suggests that targeting of CXXC5/RINF should be considered as a possible therapeutic strategy, especially in high-risk patients who show increased expression in AML cells compared with normal hematopoietic cells.

Cell division cycle 25 (CDC25) protein phosphatases regulate cell cycle progression through the activation of cyclin-dependent kinases (CDKs), but they are also involved in chromatin modulation and transcriptional regulation. CDC25 inhibition is regarded as a possible therapeutic strategy for the treatment of human malignancies, including acute myeloid leukemia (AML). We investigated the in vitro effects of CDC25 inhibitors on primary human AML cells derived from 79 unselected patients in suspension cultures. Both the previously well-characterized CDC25 inhibitor NSC95397, as well as five other inhibitors (BN82002 and the novel small molecular compounds ALX1, ALX2, ALX3, and ALX4), only exhibited antiproliferative effects for a subset of patients when tested alone. These antiproliferative effects showed associations with differences in genetic abnormalities and/or AML cell differentiation. However, the responders to CDC25 inhibition could be identified by analysis of global gene expression profiles. The differentially expressed genes were associated with the cytoskeleton, microtubules, and cell signaling. The constitutive release of 28 soluble mediators showed a wide variation among patients and this variation was maintained in the presence of CDC25 inhibition. Finally, NSC95397 had no or only minimal effects on AML cell viability. In conclusion, CDC25 inhibition has antiproliferative effects on primary human AML cells for a subset of patients, and these patients can be identified by gene expression profiling.

Background T cell mediated immune dysregulation is an established feature of Myelodysplastic syndrome (MDS). We have shown previously that the number of T regulatory cells (Tregs) are increased in high risk MDS and associate with disease progression. In low risk disease however, T helper-17 cells (Th17) are increased which correlates with augmented bone marrow apoptosis. Nevertheless, the role of other components of immune system in MDS pathogenesis is still emerging. Myeloid derived suppressor cells (MDSCs) are a group of immature myeloid cells with a potent immunosuppressive effect which are expanded in an inflammatory environment. MDSCs suppress T cells by depriving them of arginine and cysteine, as well as the secretion of Interleukin (IL)-10, Transforming Growth Factor (TGF)-β and induction of Tregs. While, the role of MDSCs in negatively regulating the anti-tumour immune responses and in determining the T cell polarity have been demonstrated in solid tumours, this information is lacking in MDS. The aim of this study was to investigate the number and function of MDSCs in MDS and potential contribution to immune dysregulation in MDS. Material and Methods MDSCs and Treg numbers were assessed in peripheral blood (PB) and bone marrow (BM). In total we have analysed PB from 24 MDS patients of whom 12 patients had concurrent BM samples. PB samples from 11 age-matched healthy donors (HD) were also included. The WHO subtypes were: RC, RARS or RCMD (n=16, 67%) and 8 patients (33%) had RAEB. Cell surface staining and flow-cytometry was performed following red cell-lysis using the following markers: Live/dead dye, (eBioscience), Lineage markers (CD3, CD16, CD19, CD20, CD56,), CD33, CD34, HLA-DR, CD11b, CD15, CD66b, CD14, CX3CR1, CXCR3 and CXCR4. MDSCs were stained for intracellular TGF-β and IL-10 following fixation and permeabilisation, and they also stained positive for Arginase-1. To evaluate MDSCs function, CFSE stained CD3+CD4+CD25- (T-effectors) from MDS patients were stimulated by anti-CD3/CD28 antibodies. Cells were cultured with: T-effectors alone, T-effectors + CD3+/CD4+/CD25high (Tregs), (ratio 2:1); T-effectors + Tregs + HLA-DR-/CD14+ (MDSC) (ratio 2:1:1). Results MDSCs in the PB were higher in patients with RAEB compared to both healthy donors (HD) and non-RAEB MDS (2.6% v 1.03% p=0.0004) & (2.6% v 2% p=0.02). The absolute numbers of MDSCs were also higher in RAEB compared to non-RAEB subtypes (0.17 x 109/L v 0.08 x 109/L, p=0.02) and in Intermediate (INTR)/High (HR)/Very high (VHR) risk groups in comparison with Very low (VLR)/ Low (LR) risk group (0.31 x 109/L v 0.08 x 109/L, p=0.01). MDSCs frequency in MDS patients was higher in BM than in PB (45% v 2.6%, p=0.002). The PB MDSCs were mainly Monocytic (M)-MDSCs (0.6 % in PB v 0.3% in BM, p=0.02). M-MDSCs were also expressing higher CX3CR1, (MFI 350 v 8849, p=0.008) and higher CXCR4 (MFI 1846 v 2498, p=0.0156) compared to G-MDSCs. Granulocytic (G)-MDSC percentages were increased in INT/HR/VHR compared to VLR/LR (0.7% v 0.2%, p=0.0089) and HD (0.2% v 0.7%, p=0.0385). G-MDSCs were also more frequent in the peripheral blood of RAEB compared to non-RAEB subtypes (0.5% v 0.1% p=0.0299). G-MDSCs from MDS patients produce higher amount of IL-10/TGF-β compared to Monocytic (M)- MDSCs, evaluated by intracellular staining (IL-10 R-MFI, 7.9 v 3.8, p=0.003 and TGF-β R-MFI 15 v 4.8, p=0.024). To evaluate the suppressive effect of MDSCs, autologous Tregs and T-effectors from MDS patients (one INTR and one LR) were co-cultured in the presence and absence of MDSCs. Presence of MDSCs, not only increased the T-effector suppression (figure 1) but also induced Tregs proliferation. In patients with RAEB we also found a positive correlation between absolute counts of MDSC and CD27+FOXP3+CD4+Tregs (Spearman R=0.9, p=0.0374). Discussion Our data show that MDSCs are increased in MDS patients, particularly in high-risk disease, compared to healthy donors. The expanded MDSCs express chemokine receptors and secrete IL-10 and TGF-β, and potentiate the suppressive effect of Tregs. In RAEB patients, numbers of MDSCs are positively correlated with Treg numbers. Data suggests that increased MDSCs play an important role in immunopathogenesis of MDS by suppressing the immune-surveillance against the dysplastic clone. Inhibition of MDSCs (i.e. by arginase inhibitor) could reverse the immunosuppressive environment and re-establish immune-surveillance in MDS. Disclosures: No relevant conflicts of interest to declare.