High Constitutive Cytokine Release by Primary Human Acute Myeloid Leukemia Cells Is Associated with a Specific Intercellular Communication Phenotype

Abstract

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.

Keywords: RAC1; SYK; acute myeloid leukemia; cytokines; differentiation; gene mutations; integrin; proteomic profile.

Figure 1

The total genomic profile and organization of mutations into defined categories; an overview of the data for the 71 AML patients included in our study. The figure shows the somatic mutations identified from a 54 gene mutation panel, the mutations being classified as described previously [6,7]. A majority of 69 patients had at least one detectable mutation. Risk classification of the karyotypes, morphological signs of differentiation (i.e., FAB-classification), etiology, age, and gender are presented in the right part of the figure. The patients selected for proteomic analyses are indexed with black in the left part of the figure.

Figure 2

The secretome and genomic profile for 46 AML patients. Primary AML cells derived from a consecutive subset of 46 patients were cultured in vitro for 48 h and the supernatant levels of 34 soluble mediators were then determined. We performed an unsupervised hierarchical cluster analysis (Euclidean measure, and complete distance) based on these results and were then able to identify two distinct patient clusters corresponding to patients with generally high or intermediate/low supernatant level.

Figure 3

Identification of two main patient subsets based on proteomic differences of AML cells with high and low constitutive release. Eight of the 16 patients included in the proteomic studies belonged to the cluster characterized by generally high constitutive mediator release and the eight others showed low/intermediate secretion (Figure 2); 256 proteins differed significantly between these two groups. We performed an unsupervised hierarchical cluster analyses (Euclidean measure, and complete distance) based on the levels of these proteins, and the left part demonstrates the dendrogram and heat map; blue indicates low protein levels and green high levels. Two main clusters were then identified corresponding to the high and low/intermediate secretion patients except for one outlier patient (left column, red color indicating high release). As expected, the two main clusters were heterogeneous with regard to mutational frequencies (middle panel) and did not differ with regard to clinical or biological characteristics either (right panel).

Figure 4

GO-terms including significantly increased proteins for AML cells with generally high constitutive release of extracellular soluble mediators. The over-representation analysis based on cellular compartment identified four GO terms with FDR < 0.05 and including at least 40 proteins, i.e., GO:0070062—extracellular exosome, GO:0005829—cytosol, GO:0016020—membrane, and GO:0005737—cytoplasm. These four GO-terms were partly overlapping (only six proteins included in all four); together they included 153 of the 186 proteins that were increased in AML cells with generally high constitutive release compared with AML cells with low/intermediate constitutive release.

Figure 5

The network analysis of proteins showing differential expression in primary AML cells with generally high versus generally low constitutive release of extracellular mediators. The intensity of the color reflects the fold change (FC) significance when comparing the high- and low-release groups; thus a negative fold change indicates increased protein abundance in the low-release group (purple) and a positive fold change indicates increased protein abundance in the high-release group (green). This STRING-DB analysis was based only on the 256 proteins that were quantified and considered significantly different between the two groups; the figure thus shows proteins from our quantified data and no shells of interactors were considered.