Acute myeloid leukemia (AML) is a hematological cancer that mainly affects the elderly. Although complete remission (CR) is achieved for the majority of the patients after induction and consolidation therapies, nearly two-thirds relapse within a short interval. Understanding biological factors that determine relapse has become of major clinical interest in AML. We utilized liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify the protein changes and protein phosphorylation events associated with AML relapse in primary cells from 41 AML patients at time of diagnosis. Patients were defined as relapse-free if they had not relapsed within a five-year clinical follow-up after AML diagnosis. Relapse was associated with increased expression of RNA processing proteins and decreased expression of V-ATPase proteins. We also observed an increase in phosphorylation events catalyzed by cyclin-dependent kinases (CDKs) and casein kinase 2 (CSK2). The biological relevance of the proteome findings was supported by cell proliferation assays using inhibitors of V-ATPase (bafilomycin), CSK2 (CX-4945), CDK4/6 (abemaciclib) and CDK2/7/9 (SNS-032). While bafilomycin preferentially inhibited the cells from relapse patients, the kinase inhibitors were less efficient in these cells. This suggests that therapy against the upregulated kinases could also target the factors inducing their upregulation rather than their activity. This study, therefore, presents markers that could help predict AML relapse and direct therapeutic strategies.

Acute myeloid leukemia (AML) is an aggressive bone marrow malignancy where the immature leukemia cells communicate with neighboring cells through constitutive cytokine release and through their cell surface adhesion molecules. The primary AML cells express various integrins. These heterodimeric molecules containing an α and a β chain are cell surface molecules that bind extracellular matrix molecules, cell surface molecules and soluble mediators. The β3 integrin (ITGB3) chain can form heterodimers only with the two α chains αIIb and αV. These integrins are among the most promiscuous and bind to a large number of ligands, including extracellular matrix molecules, cell surface molecules and soluble mediators. Recent studies suggest that the two β3 integrins are important for leukemogenesis and chemosensitivity in human AML. Firstly, αIIb and β3 are both important for adhesion of AML cells to vitronectin and fibronectin. Secondly, β3 is important for the development of murine AML and also for the homing and maintenance of the proliferation for xenografted primary human AML cells, and for maintaining a stem cell transcriptional program. These last effects seem to be mediated through Syk kinase. The β3 expression seems to be regulated by HomeboxA9 (HoxA9) and HoxA10, and the increased β3 expression then activates spleen tyrosine kinase (Syk) and thereby contributes to cytokine hypersensitivity and activation of β2 integrins. Finally, high integrin αV/β3 expression is associated with an adverse prognosis in AML and decreased sensitivity to the kinase inhibitor sorafenib; this integrin can also be essential for osteopontin-induced sorafenib resistance in AML. In the present article, we review the experimental and clinical evidence for a role of β3 integrins for leukemogenesis and chemosensitivity in AML.

Splenic tyrosine kinase (SYK) is a non-receptor tyrosine kinase. It is important for downstream signaling from several cell surface receptors, including Fc receptors, complement receptors and integrins. SYK can have either oncogenic or tumor suppressor activity in human malignancies. Recent studies suggest that SYK inhibition may have an antileukemic effect in human acute myeloid leukemia (AML). Areas covered: Relevant publications were identified through literature searches in the PubMed database. We searched for (i) original articles describing the results from clinical studies of SYK inhibition; (ii) published articles describing the importance of SYK in human malignancies, especially AML. Expert opinion: SYK is important for the downstream signaling from several cell surface receptors. There is also a crosstalk between SYK and signaling initiated through ligation of Toll like receptors, and SYK is thereby linked with the NFκB mediated transcriptional regulation. SYK activation will also influence PI3K-Akt-mTOR signaling. Several of these signaling events are important for survival and proliferation of primary human AML cells. In the present review we describe and discuss the role of SYK in human AML, and these data suggest that SYK inhibition is a possible therapeutic strategy in human AML.

Acute myeloid leukemia (AML) is an aggressive blood cancer resulting in accumulation of immature, dysfunctional blood cells in the bone marrow. Changes in cell metabolism are features of many cancers, including AML and this may be exploited as a therapeutic target. In this study we investigated the in vitro antileukemic effects of seven metabolic inhibitors that target different metabolic pathways. The metabolic inhibitors were tested on AML cells derived from 81 patients using proliferation and viability assays; we also compared global gene expression and proteomic profiles for various patient subsets. Metformin, 2DG, 6AN, BPTES and ST1326 had strong antiproliferative and proapoptotic effects for most patients, whereas lonidamine and AZD3965 had an effect only for a minority. Antiproliferative effects on AML cells were additive when combined with the chemotherapeutic agent AraC. Using unsupervised hierarchical clustering, we identified a strong antiproliferative effect on AML cells after treatment with metabolic inhibitors for a subset of 29 patients. Gene expression and proteomic studies suggested that this subset was characterized by altered metabolic and transcriptional regulation. In addition, the Bcl-2 inhibitor venetoclax, in combination with 2DG or 6AN, increased the antiproliferative effects of these metabolic inhibitors on AML cells. Therapeutic targeting of cellular metabolism may have potential in AML, but the optimal strategy will likely differ between patients.

Acute myeloid leukemia (AML) is a heterogeneous disease, and this heterogeneity includes the capacity of constitutive release of extracellular soluble mediators by AML cells. We investigated whether this capacity is associated with molecular genetic abnormalities, and we compared the proteomic profiles of AML cells with high and low release. AML cells were derived from 71 consecutive patients that showed an expected frequency of cytogenetic and molecular genetic abnormalities. The constitutive extracellular release of 34 soluble mediators (CCL and CXCL chemokines, interleukins, proteases, and protease regulators) was investigated for an unselected subset of 62 patients, and they could be classified into high/intermediate/low release subsets based on their general capacity of constitutive secretion. FLT3-ITD was more frequent among patients with high constitutive mediator release, but our present study showed no additional associations between the capacity of constitutive release and 53 other molecular genetic abnormalities. We compared the proteomic profiles of two contrasting patient subsets showing either generally high or low constitutive release. A network analysis among cells with high release levels demonstrated high expression of intracellular proteins interacting with integrins, RAC1, and SYK signaling. In contrast, cells with low release showed high expression of several transcriptional regulators. We conclude that AML cell capacity of constitutive mediator release is characterized by different expression of potential intracellular therapeutic targets.

word count: 213 Number of figures and tables: 5 Number of references: 84 Key Points  V-ATPases and RNA processing proteins are downregulated and upregulated, respectively, in relapse patients  Relapse patients show higher activity of CSK2 and CDKs when compared to relapse-free patients AbstractAcute myeloid leukemia (AML) is a hematological cancer that mainly affects the elderly.Although complete remission (CR) is achieved for majority of the patients after induction and consolidation therapies, nearly two-thirds relapse within a short interval. Understanding biological factors that determine relapse has therefore become of major clinical interest in AML.We utilized liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify protein changes and protein phosphorylation events associated with AML relapse in primary cells from 41 AML patients at time of diagnosis. Patients were defined as relapse-free if they had not relapsed within a 5-year clinical follow-up after AML diagnosis. Relapse was associated with increased expression of RNA processing proteins and decreased expression of V-ATPase proteins. We also observed an increase in phosphorylation events catalyzed by cyclin-dependent kinases (CDKs) and casein kinase 2 (CSK2). The biological relevance of the proteome findings was supported by cell proliferation assays using inhibitors of V-ATPase (bafilomycin), CSK2 (CX-4945), CDK4/6 (abemaciclib) and CDK2/7/9 (SNS-032). While bafilomycin preferentially inhibited the cells from relapse patients, the kinase inhibitors were less efficient in these cells.This suggests that therapy against the upregulated kinases also could target the factors inducing their upregulation rather than their activity. In conclusion, our study presents markers that could help predict AML relapse and direct therapeutic strategies.

The constitutive PI3K-Akt-mTOR activation differed between patients; this difference appears to be a part of complex phenotypic differences including cell communication, intracellular signaling through other pathways, and transcriptional regulation.

Hypoxia is one of the factors that trigger the release of inflammatory and vasculogenic cytokines during tissue regeneration. Mesenchymal stem cells (MSC) with and without endothelial cells (EC) were cultured in vitro in normoxic and hypoxic environments. The mRNA expression of inflammatory and vasculogenic cytokines were evaluated at 1, 12, 24 and 48 hours. After 48 hours of incubation in normoxic and hypoxic conditions, supernatants termed as conditioned medium (CM) from each group were collected and analyzed. The protein level of VEGF-A in the CM was determined by ELISA. The effects of the CM from different groups on EC were evaluated using wound healing-and tube formation assays. The mRNA expression of IL-1β, IL-6 and IL-8 was up-regulated in both the normoxic and hypoxic co-culture (MSC/EC) group, compared with the mono-culture normoxic group. The VEGF-A protein level was higher in the hypoxic mono-and co-culture group. Wound closure was accelerated by CM from the mono-and co-culture hypoxic groups compared with both normoxic groups. Measurements of tube formation were higher in the hypoxic mono-culture group compared with the normoxic group. The conditioned medium obtained from hypoxia preconditioning of the cells accelerated wound healing and vessel formation in vitro.