Acute myeloid leukemia (AML) remains a devastating disease. This is mainly due to limited treatment options for patients with relapsed or refractory disease and those with FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations. FLT3-ITD occurs in approximately a quarter of patients with AML and confers poor prognosis due to high propensity for relapse after remission. The discovery of novel therapies is necessary for cure of AML. Pateamine A (Pat A) is a natural protein translation inhibitor that targets the eukaryotic initiation factor 4A (eIF4A) and inhibits 5'CAP dependent translation. A previous study by Galina Kuznetsov revealed that des-methyl, des-amino Pateamine A (DMDA-Pat A), a synthetic analogue of Pat A, had potent anti-proliferative activity against several human cancer cell lines, including melanoma, colon cancer, non-small cell lung cancer and pre-B acute lymphoid leukemia (Molecular Cancer Therapeutics 2009). The effectiveness of Pat A on AML cells has yet to be elucidated. The primary objective of this study was to determine if Pat A decreased AML FLT3-ITD cell proliferation and caused cell death. The secondary objective was to determine if Pat A decreased AML FLT3-ITD cell protein translation. Lastly, the tertiary objectives were to compare Pat A to AML therapies currently under investigation and in clinical practice and to examine the effects of combination treatments with Pat A on AML FLT3-ITD cells.
Human acute myeloid leukemia MV411 (FLT3-ITD) cells were seeded in the presence of increasing concentrations of DMDA-Pat A. After 48 hours cell proliferation was assessed using MTS assay. To determine if MV411 cells underwent cell death, western blot analysis for PARP-cleavage and fluorometric caspase activation analysis were performed on cell lysates after DMDA -Pat A treatment for 24 hours. Western blot analysis for cell survival regulatory proteins, XIAP, MCL-1, BCL2 and BCL-XL, were performed on treated cell lysates as well. To determine if DMDA-Pat A decreased MV411 protein translation, cells were treated for 3 hours with DMDA-Pat A and labeled with 35S-methionine during last 15 minutes of experiment. Radiolabeled proteins were trichloroacetic acid (TCA) precipitated and measured by scintillation counting. In order to compare DMDA-Pat A to compounds currently in clinical practice, MTS cell proliferation assays were performed on MV411 cells with increasing concentrations of Idarubicin, Cytarabine and Midostaurin (PKC 412; FLT3 inhibitor). Also DMDA-Pat A was compared to Torin, a mammalian target of rapamycin complex (mTORC1/2) inhibitor, which is currently under investigation for treatment of AML. Lastly, MV411 cells were treated with DMDA-Pat A in combination with the above compounds in order to possibly produce synergistic inhibition of cell growth.
DMDA-Pat A decreased MV411 cell proliferation with an IC50 of approximately 20 nM. The maximum inhibition of cell growth was achieved at 100nM of DMDA-Pat A. Western blot analyses revealed that MV411 cells underwent cell death as determined by PARP cleavage with 20nM and 100nM of DMDA-Pat A. Fluorometric caspase activation assay revealed an increase in caspase activation with 20nM DMDA-Pat A treatment. Cell survival regulatory proteins XIAP, MCL-1 and BCL-XL were all decreased whereas BCL-2 was unchanged. In comparison to control, MV411 protein translation was decreased by 50% after 20 nM DMDA-Pat A treatment. Idarubicin, Cytarabine, Midostaurin and Torin were all able to decrease MV411 cell proliferation, but the half maximal inhibitory concentrations (IC50) of all four compounds were greater than DMDA-Pat A (Idarubicin IC50 100nm, Cytarabine IC50 30nm, Midostaurin IC50 125nm and Torin IC50 60 nm). Lastly, there was no synergistic inhibition of cell growth with DMDA-Pat A and any of the above four compounds, but DMDA-Pat A in combination with Idarubicin produced additive inhibition of cell growth.
DMDA-Pat A decreases proliferation of FLT3-ITD MV411 cells by reducing cell survival proteins and inducing apoptosis at low concentrations. DMDA-Pat A was determined to be effective at concentrations lower than another protein synthesis inhibitor (Torin) or other cytotoxic agents (Idarubicin, Cytarabine, Midostaurin). In combination with other cytotoxic drugs, DMDA-Pat A could be a potential treatment option for AML patients with FLT3-ITD mutations.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.