Pharmacologic Doses of Amiloride Preferentially Induce Apoptosis and Growth Inhibition of Flt3-ITD Mutation Positive Acute Myeloid Leukemia Cell Lines

Amiloride is an FDA approved potassium-sparing diuretic which targets Na+/H+ exchanger isoform 1 (NHE1). NHE1 is responsible for the regulation of the intracellular pH, as well as cell-cycle and apoptosis. In supra-pharmacologic concentrations, amiloride non-specifically inhibits protein kinases. Recent study demonstrated that proapoptotic effect of amiloride in CML cell lines is linked to the modulation of the alternative splicing of Bcl-x, HIPK3, and BCR/ABL genes and is independent of pHi.

Here, we demonstrate that pharmacologic doses of amiloride preferentially induce growth inhibition, cell cycle arrest and apoptosis in Flt3-ITD positive acute myeloid leukemia cell lines as compared to Bcr-Abl positive leukemia cell line. Our data suggests that amiloride may have an effect on Flt3 signaling and that its treatment potential for Flt3-ITD positive acute myeloid leukemia needs to be explored.

MV4-11, MOLM13 and K562 cells lines in log-phase growth were used for the experiments. Analysis of the baseline Flt3 expression and phosphorylation status was assessed via Flt3 immunoprecipitation and Western blotting for Flt3 and phosphotyrosine. Cells were incubated with various amiloride concentrations; equal volume dilutions of DMSO were used for control. Cell counting and trypan blue exclusion viability was performed on TC10 Bio-Rad automated cell counter. The cell cycle analysis was performed applying propidium iodide staining. To assess for apoptosis and cell death, we used annexin V/PI staining kit and flow cytometry.

MOLM13 and MV4-11 cell lines carry activating Flt3-ITD mutation. We confirmed the expression and constituative activation of Flt3 in MOLM13 and MV4-11 cells with Western blotting. Flt3 protein was not detectable in K562 cell line. Amiloride at 0.025 mM and 0.05 mM completely inhibited the growth of MV4-11 cells after 24 hrs of treatment with no significant increase in total or live cell numbers at 72 hrs, but only mildly affected K562 cell proliferation. While the above amiloride concentrations caused cell death in MV4-11 and MOLM13 cell lines, there was no increased cell death in K562 cells.

Incubation of MOLM13 and MV4-11 cell lines with 0.05 mM amiloride for 20 hrs induced cell cycle arrest. In MV4-11 cell line, the proportion of S phase cells after amiloride treatment was 15.4% (SD=5.4%) as compared to 31.3% (SD=1.4%) in control. MOLM13 cell line demonstrated 15.3% (SD=4.7%) of cells in S after amiloride treatment as compared to 35.3% (SD=2.4%) cells in S phase in control treatment. In K562 cell line, there was less effect with 52% (SD=4.2%) of cells in S phase in control as compared to 37% (SD=8.9%) in amiloride treatment.

MV4-11 and MOLM 13 cell lines were more sensitive than K562 cells to amiloride induced apoptosis with 28.8% (control 12.7%) of MV4-11 cells, 11.4% (control 7.4%) of MOLM13 cells, and 11.4% (control 8.6%) of K562 cells being apoptotic after 20 hr treatment with 0.05mM amiloride. At 72 hrs of amiloride treatment 34% (control 1.5%) of MV4-11 cells, 17% (control 5%) of MOLM13 cells and 11% of K562 cells (control 8.9%) were apoptotic. Amiloride had similar effect on the number of dead cells with no increase in total cell death in K562 cell line. Upon treatment with increasing amiloride concentrations, there was dose-dependent increase in cell death and apoptosis in all three cell lines with K562 line showing relative resistance to amiloride.

Our results demonstrate that amiloride induces cell cycle arrest and inhibits proliferation of Flt3-ITD positive cell lines MV4-11 and MOLM13 as well as K562 cell line at a pharmacologic concentration of 0.05 mM. Both, cell cycle arrest and antiproliferative effect are more pronounced in Flt3-ITD positive cells lines while it is mild in Bcr-Abl positive K562 cell line. Pharmacologic doses of amiloride induce cell death and apoptosis in Flt3-ITD positive cell lines but not in K562 cell line. Both, Bcr-Abl and Flt3 signaling stimulates proliferation and inhibits apoptosis in myeloid leukemia cells. Our study suggests that amiloride may induce cell cycle arrest and apoptosis via modulating Flt3 signaling cascade. We are currently investigating the effects of amiloride on Flt3 phosphorylation. In conclusion, our data suggests that amiloride presents a potential treatment option for Flt3-ITD positive acute myeloid leukemia.

No relevant conflicts of interest to declare.