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. 2017 Mar 11;22(3):446.
doi: 10.3390/molecules22030446.

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

Annette K Brenner  1 , Håkon Reikvam  2 , Kristin Paulsen Rye  3 , Karen Marie Hagen  4 , Antonio Lavecchia  5 , Øystein Bruserud  6   7
Affiliations

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

Annette K Brenner et al. Molecules. .

Abstract

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.

Keywords: CDC25 inhibitors; acute myeloid leukemia; cytogenetics; gene expression; leukemic cell differentiation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Chemical structures of cell division cycle 25 (CDC25) inhibitors used in the study: 5-((2-hydroxy-4-methylquinolin-6-yl)methyl)-2-methoxybenzenesulfonic acid (ALX1), 5,5′-(3-(pyridin-2-yl)-1,2,4-triazine-5,6-diyl)-difuran-2-sulfonic acid (ALX2), 4-(2-carboxybenzoyl)-phthalic acid (ALX3), 2,6,7-trihydroxy-9-phenyl-3H-xanthen-3-one (ALX4), 2,3-bis-((2-hydroxyethyl)thio)-1,4-naphthoquinone (NSC95397) and 4-dimethylamino-2-methoxy-6-((methyl-(2-(4-nitrophenyl)ethyl)amino)methyl)phenol (BN82002).
Figure 2
Figure 2
Effects of CDC25 inhibitors on cytokine-dependent (granulocyte macrophage colony-stimulating factor, stem cell factor and FMS-related tyrosine kinase 3 ligand) AML cell proliferation. Primary AML cells were cultured in suspension cultures prepared with medium alone or in medium supplemented with ALX4, at a final concentration of 10 μM. Cytokine-dependent AML cell proliferation was assayed as (3H)-thymidine incorporation after seven days of culture (i.e., (3H)-thymidine added on day six); a proliferation at this time after the initiation of culture reflects an enrichment of clonogenic cells [42]. The results are presented as the nuclear incorporation of radioactivity (counts per minute, cpm), and the figure shows box plots (median and quartiles), together with the 90% confidence intervals and outliers. All statistically significant differences are indicated at the top of the figures. (a) The effect of CDC25 inhibition on AML cell proliferation was analyzed for all AML cells (left), cells with non-favorable cytogenetics (middle), and cells with favorable cytogenetics (right); for each of these three comparisons, the results for ALX4 (white), NSC95397 (light grey), and drug-free control cultures (dark grey) are presented; (b) The effect of ALX4 on AML cell proliferation was also compared for all patients and patients with CD34+/CD34− leukemic cells. The figure compares the results for cultures prepared with (white) and without ALX4 (grey); (c) The effect of ALX4 on AML cell proliferation, a comparison of all patients, patients showing no/few morphological signs of differentiation (corresponding to FAB-M0/1), and patients showing monocytic differentiation. For each of these three analyses, the figure presents the results for cultures with (white) and without ALX4 (grey).
Figure 3
Figure 3
Comparison of gene expression profiles for primary human AML cells—a comparison of AML cell populations with and without an antiproliferative effect of CDC25 inhibitors. (LEFT) Cells derived from 62 AML patients were cultured in suspension cultures ((3H)-thymidine incorporation assay), in combination with six different CDC25 inhibitors (ALX1-4, BN82002 and NSC95397). Only patients without the t(15;17) cytogenetic aberration and with proliferation above >1000 cpm, both in control and drug-containing cultures, were included in this analysis. The (3H)-thymidine incorporation (cpm) for drug-containing cultures was made relative to the incorporation for corresponding drug-free controls and thereafter log2 converted. These values were then used for an unsupervised hierarchical cluster analysis (weighted pair group method with averaging, Euclidian distance measure). Green color represents an inhibitory and thus antiproliferative effect. (MIDDLE) Based on this analysis, the patients could be divided into two main subsets, as indicated by the bar between the two main parts of the figure; responders showing antiproliferative effects (green part of the column) and non-responders with no antiproliferative effect and even showing growth enhancement (red part of the column) in the presence of CDC25 inhibitors. (RIGHT) Global gene expression profiles were available for 39 unselected patients (16 with antiproliferative and 23 without antiproliferative effects of CDC25 inhibition). Patients with and without antiproliferative effects were compared by a feature subset analysis, and 87 differentially expressed genes (p < 0.003) were detected. These 87 genes could be used for a hierarchical cluster analysis (weighted pair group method with averaging, Euclidian distance measure) that divided the patients into two main subsets, corresponding to the 16 patients with and the 23 patients without an antiproliferative effect induced by CDC25 inhibitors. Green indicates upregulated genes and red indicates downregulated genes.
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