The potassium channel Ether à go-go is a novel prognostic factor with functional relevance in acute myeloid leukemia

Abstract

Background: The voltage-gated potassium channel hEag1 (KV10.1) has been related to cancer biology. The physiological expression of the human channel is restricted to the brain but it is frequently and abundantly expressed in many solid tumors, thereby making it a promising target for a specific diagnosis and therapy. Because chronic lymphatic leukemia has been described not to express hEag1, it has been assumed that the channel is not expressed in hematopoietic neoplasms in general.

Results: Here we show that this assumption is not correct, because the channel is up-regulated in myelodysplastic syndromes, chronic myeloid leukemia and almost half of the tested acute myeloid leukemias in a subtype-dependent fashion. Most interestingly, channel expression strongly correlated with increasing age, higher relapse rates and a significantly shorter overall survival. Multivariate Cox regression analysis revealed hEag1 expression levels in AML as an independent predictive factor for reduced disease-free and overall survival; such an association had not been reported before. As a functional correlate, specific hEag1 blockade inhibited the proliferation and migration of several AML cell lines and primary cultured AML cells in vitro.

Conclusion: Our observations implicate hEag1 as novel target for diagnostic, prognostic and/or therapeutic approaches in AML.

Figure 1

hEag1 expression patterns in leukemia. A. Percentage of hEag1 positive patients as a function of cytological diagnosis. The percentage of hEag1 positive samples in each subgroup is given on top of each bar pair. hEag1 expression was subtype dependent in AML.

Figure 2

Expression of hEag1 in leukemia cell lines and primary AML cells. A: hEag1 mRNA levels determined by real-time PCR, expressed as relative to that in UT-7 cells. B: hEag1 protein content in cell lines and primary cells from hEag1 positive patients measured by flow cytometry. The background rightward shift induced by anti-hEag1 antibody mAb49 in the PCR-negative P2 cells was set as zero value. C: hEag1 siRNA treatment (lower panels) strongly diminished the fluorescence shift attributable to hEag1 expression in PLB-985 and K562 cells. The gray peak indicates control secondary fluorescent antibody staining.

Figure 3

hEag1 expression correlated to three age groups: 0-18, 19-59 and 60-89 years. With increasing age hEag1 expression became more abundant.

Figure 4

Shorter survival in hEag1 positive patients. Kaplan-Meier curves show the disease-free and overall survival of patients expressing (black line) or not expressing (grey dashed line) hEag1. A significantly shorter disease-free (7 vs. 22 months, p = 0.0023) and overall survival (10 vs. 52 months, p = 0.0019) can be observed in hEag1 positive versus hEag1 negative patients.

Figure 5

Effects of hEag1 blockade on proliferation of leukemia cells in vitro. Proliferation inhibition of PLB-985 (A), UT-7 (B), K562 (C) and primary cells (P4; D) treated with 20 μM imipramine (filled triangles), 4 μM astemizole (open triangles), 10 μg/mL monoclonal hEag1 antibody mAb56 (filled circles) or 30 nM anti-hEag1 siRNA (open squares) is shown over 4 or 5 days as compared to control cells (open circles). (Mean ± SEM).

Figure 6

Cell cycle distribution and apoptosis in the presence of hEag1 inhibitors in vitro. A. The indicated cell lines were incubated with 4 μM astemizole (AST), 20 μM imipramine (IMI) or 10 μg/mL anti-hEag1 mAb56 for 2 days. Cell cycle phases were measured after staining with propidium iodide by flow cytometry. Only mAb56 increased significantly the proportion of cells in the S phase in K562 and HEL cells. B. K-562 and primary P4 cells were incubated with 4 μM astemizole or mAb56 antibody for 2 days, respectively and apoptosis was measured by flow cytometry. A clear increase in Annexin V fluorescence (apoptosis) was observed in both determinations.

Figure 7

Induction of apoptosis by chemotherapeutic drugs. Apoptosis was measured through caspase activation and is represented as relative increase with respect to basal apoptosis in PLB-985 (A), K562 (B) and primary cultured P4 cells (C). Significant increases were observed in PLB-985 cells. In this cell type, Astemizole and mAb56 increased the efficacy of the drugs.

Figure 8

Inhibition of the migration of cell lines in the presence of hEag1 inhibitors. HEL and PLB-985 cell migration was reduced by the specific anti-hEag1 mAb56.