Heterogeneity of Patient-Derived Acute Myeloid Leukemia Cells Subjected to SYK In Vitro Inhibition.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy with a dismal prognosis. The cytoplasmic spleen tyrosine kinase (SYK) is highly expressed by hematopoietic cells and has emerged as a potential therapeutic target. In this study, we evaluated the in vitro antileukemic effects of five SYK inhibitors, fostamatinib, entospletinib, cerdulatinib, TAK-659, and RO9021, in a consecutive AML patient cohort. All inhibitors demonstrated a concentration-dependent antiproliferative effect, although there was considerable heterogeneity among patients. For fostamatinib and TAK-659, the antiproliferative effects were significantly higher in FLT3 mutated patients compared to nonmutated patients. Fostamatinib, entospletinib, TAK-659, and RO9021 induced significant apoptosis in primary AML cells, although the proapoptotic effects of the SYK inhibitors were less pronounced than the antiproliferative effects. Finally, most of the SYK inhibitors caused a significant decrease in the release of cytokines and chemokines from primary AML cells, indicating a potent inhibitory effect on the release of these leukemic signaling molecules. We concluded that the SYK inhibitors had antileukemic effects in AML, although larger studies are strongly needed to identify which patient subsets will benefit most from such a treatment.

Giardia-specific cellular immune responses in post-giardiasis chronic fatigue syndrome.

BACKGROUND: The role of pathogen specific cellular immune responses against the eliciting pathogen in development of post-infectious chronic fatigue syndrome (PI-CFS) is not known and such studies are difficult to perform. The aim of this study was to evaluate specific anti-Giardia cellular immunity in cases that developed CFS after Giardia infection compared to cases that recovered well. Patients reporting chronic fatigue in a questionnaire study three years after a Giardia outbreak were clinically evaluated five years after the outbreak and grouped according to Fukuda criteria for CFS and idiopathic chronic fatigue. Giardia specific immune responses were evaluated in 39 of these patients by proliferation assay, T cell activation and cytokine release analysis. 20 Giardia exposed non-fatigued individuals and 10 healthy unexposed individuals were recruited as controls. RESULTS: Patients were clinically classified into CFS (n = 15), idiopathic chronic fatigue (n = 5), fatigue from other causes (n = 9) and recovered from fatigue (n = 10). There were statistically significant antigen specific differences between these Giardia exposed groups and unexposed controls. However, we did not find differences between the Giardia exposed fatigue classification groups with regard to CD4 T cell activation, proliferation or cytokine levels in 6 days cultured PBMCs. Interestingly, sCD40L was increased in patients with PI-CFS and other persons with fatigue after Giardia infection compared to the non-fatigued group, and correlated well with fatigue levels at the time of sampling. CONCLUSION: Our data show antigen specific cellular immune responses in the groups previously exposed to Giardia and increased sCD40L in fatigued patients.

CDC25 Inhibition in Acute Myeloid Leukemia-A Study of Patient Heterogeneity and the Effects of Different Inhibitors.

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.

The Systemic Profile of Soluble Immune Mediators in Patients with Myelodysplastic Syndromes.

INTRODUCTION: Myelodysplastic syndromes (MDS) are characterized by bone marrow failure due to disturbed bone marrow maturation. MDS is associated with increased risk of transformation to acute myeloid leukemia (AML) and features of immunological dysregulation. MATERIALS AND METHODS: Serum levels of 47 soluble immune mediators were examined in samples derived from 49 MDS patients (35 low-risk and 14 high-risk) and 23 healthy adults. Our patients represent an unselected population-based cohort. The mediators included cytokines, soluble adhesion proteins, matrix metalloproteases, and tissue inhibitors of proteases. Levels were determined using Luminex assays. Patients were classified as low- and high-risk based on the international prognostic scoring system (IPSS) score. RESULTS: When comparing the serum levels of single mediators the MDS patients showed a relatively wide variation range for several mediators compared with healthy adults, especially interleukin 6 (IL-6), IL-8/CXCL8, CCL3, and CCL4. The high-risk patients had lower levels of epidermal growth factor (EGF), cluster of differentiation 40 ligand (CD40L), CCL5, CCL11, CXCL5, matrix metalloproteinase 1 (MMP-1), MMP-9, and tissue inhibitor of metalloproteinases 2 (TIMP-2) compared with low-risk patients. Unsupervised hierarchical cluster analysis visualized marked serum mediator profile differences between MDS patients; based on this analysis three patient subsets could be identified. The healthy adults were also included in this analysis and, as expected, they formed their own separate cluster, except for one outlier. Both low- and high-risk patients showed considerable heterogeneity with regard to serum profile, and this heterogeneity seems stable over time (one year follow-up). Finally, very few mediators differed between low- and high-risk patients, but hierarchical clustering based both on all mediators, as well as five selected mediators (EGF, CCL11, TIMP-2, MMP-1, and MMP-9) identified subsets of patients with significantly increased frequency of high-risk disease (χ-square test p = 0.0158 and p = 0.0148).

Pretransplant Levels of CRP and Interleukin-6 Family Cytokines; Effects on Outcome after Allogeneic Stem Cell Transplantation.

Several pretransplant factors, including CRP (C-reactive protein) levels, reflect the risk of complications after allogeneic stem cell transplantation. IL-6 induces CRP increase, and we therefore investigated the effects of pretransplant IL-6, soluble IL-6 receptors, IL-6 family cytokines and CRP serum levels on outcome for 100 consecutive allotransplant recipients. All patients had related donors, none had active infections and 99 patients were in complete remission before conditioning. The incidence of acute graft versus host disease (aGVHD) requiring treatment was 40%, survival at Day +100 82%, and overall survival 48%. Despite a significant correlation between pretransplant CRP and IL-6 levels, only CRP levels significantly influenced transplant-related mortality (TRM). However, CRP did not influence overall survival (OS). Pretransplant IL-31 influenced late TRM. Finally, there was a significant association between pretransplant IL-6 and early postconditioning weight gain (i.e., fluid retention), and this fluid retention was a risk factor for aGVHD, TRM and OS. To conclude, pretransplant CRP, IL-31 and early posttransplant fluid retention were independent risk factors for TRM and survival after allotransplantation.

Vacuolar ATPase Is a Possible Therapeutic Target in Acute Myeloid Leukemia: Focus on Patient Heterogeneity and Treatment Toxicity.

Vacuolar ATPase (V-ATPase) is regarded as a possible target in cancer treatment. It is expressed in primary acute myeloid leukemia cells (AML), but the expression varies between patients and is highest for patients with a favorable prognosis after intensive chemotherapy. We therefore investigated the functional effects of two V-ATPase inhibitors (bafilomycin A1, concanamycin A) for primary AML cells derived from 80 consecutive patients. The V-ATPase inhibitors showed dose-dependent antiproliferative and proapoptotic effects that varied considerably between patients. A proteomic comparison of primary AML cells showing weak versus strong antiproliferative effects of V-ATPase inhibition showed a differential expression of proteins involved in intracellular transport/cytoskeleton functions, and an equivalent phosphoproteomic comparison showed a differential expression of proteins that regulate RNA processing/function together with increased activity of casein kinase 2. Patients with secondary AML, i.e., a heterogeneous subset with generally adverse prognosis and previous cytotoxic therapy, myeloproliferative neoplasia or myelodysplastic syndrome, were characterized by a strong antiproliferative effect of V-ATPase inhibition and also by a specific mRNA expression profile of V-ATPase interactome proteins. Furthermore, the V-ATPase inhibition altered the constitutive extracellular release of several soluble mediators (e.g., chemokines, interleukins, proteases, protease inhibitors), and increased mediator levels in the presence of AML-supporting bone marrow mesenchymal stem cells was then observed, especially for patients with secondary AML. Finally, animal studies suggested that the V-ATPase inhibitor bafilomycin had limited toxicity, even when combined with cytarabine. To conclude, V-ATPase inhibition has antileukemic effects in AML, but this effect varies between patients.

NPM1-Mutated Patient-Derived AML Cells Are More Vulnerable to Rac1 Inhibition.

The prognosis of acute myeloid leukemia (AML) is poor, especially for the elderly population. Targeted therapy with small molecules may be a potential strategy to overcome chemoresistance and improve survival in AML. We investigated the inhibition of the signaling molecule ras-related C3 botulinum toxin substrate 1 (Rac1) in leukemia cells derived from 79 consecutive AML patients, using five Rac1 inhibitors: ZINC69391, ITX3, EHOP-016, 1A-116, and NSC23766. In vitro cell proliferation and apoptosis assays and the assessment of cytokine profiles in culture media were conducted. All five inhibitors had an antiproliferative effect; IC50 ranged from 3−24 µM. They induced significant apoptosis and necrosis compared to the untreated controls (p < 0.0001) at concentrations around IC40 and IC80. A high versus an intermediate or low antiproliferative effect was more common in NPM1-mutated (p = 0.002) and CD34-negative (p = 0.008) samples, and when NPM1 and FLT3 (p = 0.027) were combined. Presence of NPM1 mutation was associated with reduced viability after treatment with EHOP-016 (p = 0.014), ITX3 (p = 0.047), and NSC23766 (p = 0.003). Several cytokines crucial for leukemogenesis were reduced after culture, with the strongest effects observed for 1A-116 and NSC23766. Our findings suggest potent effects of Rac1 inhibition in primary AML cells and, interestingly, samples harboring NPM1 mutation seem more vulnerable.

Effects of insulin and pathway inhibitors on the PI3K-Akt-mTOR phosphorylation profile in acute myeloid leukemia cells.

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.

Expansion of myeloid derived suppressor cells correlates with number of T regulatory cells and disease progression in myelodysplastic syndrome.

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.

The importance of sample collection when using single cytokine levels and systemic cytokine profiles as biomarkers--a comparative study of serum versus plasma samples.

BACKGROUND: Cytokines, soluble adhesion molecules and metalloproteinases can be detected in human serum or plasma samples. Such systemic levels are widely used as biomarkers in epidemiological and clinical studies. METHODS: We prepared serum samples and three types of plasma samples (EDTA, heparin, citric acid) from 20 healthy individuals. The levels of 31 cytokines, four soluble adhesion molecules and eight matrix metalloproteinases were analyzed by Luminex technology. RESULTS: Most mediators showed detectable levels in both plasma and serum. Several mediators that can be released by platelets showed increased serum levels, especially CCL5 and CD40L, but for the other mediators the serum levels did not correlate with peripheral blood platelet counts and for these last mediators serum and plasma levels often showed strong correlations. The use of bivalirudin for anticoagulation significantly increased and citric acid combined with platelet inhibitors (ticagrelor, acetylsalicylic acid plus prostaglandin E2) did not alter plasma levels of platelet-store mediators compared with citric acid alone. The impact of sample preparation differed between mediators; for many mediators strong correlations were seen between serum and plasma levels even when absolute levels differed. Soluble adhesion molecule levels showed only minor differences between samples. Unsupervised hierarchical clustering suggested that the effect of sampling/preparation was strongest for serum and heparin plasma samples. CONCLUSION: Careful standardization of sample preparation is usually necessary when analyzing systemic mediator levels, and differences caused by sample preparation should be considered as a possible explanation if studies show conflicting results.

Expression patterns of chemokine receptors on circulating T cells from myelodysplastic syndrome patients.

Chemokines and their receptors are involved in the recruitment of leukocytes to sites of inflammation. Recently, chemokine expression signatures have been reported to convey a prognostic value in myelodysplastic syndrome (MDS) patients. In the present study, we investigated the chemokine receptor repertoire on fresh peripheral blood lymphocytes from 31 (22 low-risk and 9 high-risk) patients affected by MDS. Chemokine receptor expression was studied in defined T-cell subsets using eight-color flow cytometry. MDS patients exhibited quantitative differences in peripheral lymphocyte subpopulations. In addition, T cells obtained from MDS patients expressed a chemokine receptor pattern suggesting a dominance of mature and activated T cells. This is illustrated by increased levels of CCR3, CCR5, CX3CR1 and/or by a decreased abundance of CCR7 in defined T-cell subsets. The T-cell subset distribution appears to differ between the peripheral blood and the bone marrow of MDS patients, suggesting a preferential recruitment of specific T-cell subsets to the latter compartment. Alteration in chemokine receptor expression can develop over time even in patients that are considered clinically stable. Elevated expression levels of CXCR4 by CD8+ cells were associated with prolonged patient survival and reduced numbers of bone marrow blasts. We conclude that immunological abnormalities in MDS also involve chemokine receptors on different subsets of T cells, and that these changes may have a prognostic value.