Abstract

Background

Outcomes of treatment for AML are suboptimal with ara-C and anthracyclines particularly in AML with internal tandem duplication (ITD) mutation of the fms-like tyrosine kinase 3 (FLT3-ITD), which is known for hyperproliferative state with activation of glycolytic metabolism and increased intracellular reactive oxygen species (ROS). ATO is highly effective in treatment of acute promyelocytic leukemia (APL). In addition to degradation of PML-RARα in APL, ATO has multiple targets within the cell including reduction of phosphorylation of STAT proteins (downstream of FLT3) and decreasing mitochondrial membrane potential leading to apoptosis. By inhibiting pyruvate dehydrogenase kinase, DCA is known to shunt pyruvate away from lactate production and into mitochondrial respiration, resulting in enhanced oxidative phosphorylation. DCA has been used clinically for treatment of lactic acidosis in children and adults. We hypothesized that treating leukemic cells, particularly with FLT3-ITD mutations, for short periods of time with sublethal dose of DCA (priming) might thus activate mitochondria to produce more ROS on exposure to ATO, resulting in enhanced anti-leukemic activity.

Methods

IC50s for ATO and DCA were generated in AML cell lines with FLT3-ITD (MOLM14, and MV4-11), with FLT3 point mutation (MonoMac6), and with FLT3-WT (THP-1), and normal karyotype FLT3-WT primary AML cells (AML13, AML15, AML16). To generate potentiation factors, cells were primed with IC30 of DCA or vehicle for 48 h and then treated with a range of ATO concentrations in the presence or absence of DCA (IC30) for an additional 48 h. Primary cells were treated with DCA or vehicle for 24 h and then the combination for 24 h. Each experiment was terminated with WST1 (Roche). Cell viability assays were carried out similarly except the endpoint was trypan blue exclusion. Combination Index (CI) was calculated by the fixed ratio method of Chou and Talalay. Apoptosis was monitored using the FITC Annexin V Kit (BD Pharmingen) while mitochondrial potential was assessed by MitoRed Kit (Millipore); cells were analyzed by FACScan (BD Biosciences) and Flow Jo Software (Tree Flo). Numbers are mean ± SD (triplicates).

Results

IC50s of ATO and DCA alone, and when ATO is combined sequentially with IC30 of DCA are shown in Table 1. Presence of DCA increased the cytotoxicity of ATO by 1.3- to 2.2-fold (potentiation factor), and decreased cell viability by approximately 45-95% only in cell lines with FLT3 mutations (MOLM-14, MV4-11, MonoMac6). Cytotoxicity of ATO in FLT3-WT cells, THP-1, was not potentiated with DCA because cells were highly sensitive to DCA alone. A clear synergistic, but not additive, anti-leukemic effect (CI< 1.0) was observed with DCA and ATO in FLT3 mutated cell lines. When MOLM-14 cells were primed with DCA for 24 h and treated with ATO at IC50 for 48 h, an 83% increase in apoptosis (p=0.002), and a 55% increase in loss of mitochondrial membrane potential (p=0.003) were observed compared to no priming. With no DCA priming and combination of ATO+DCA for 48 h, apoptosis and loss of mitochondrial membrane potential were increased by 50% (p= 0.003), and 120% (p=0.008), respectively, compared to ATO alone. To a lower extent, the same effect was observed in primary AML cells.

Table
IC50IC50 of ATO primed with DCA vs IC50 of ATO alone (PF)Decreased Cell Viability (%)*CI (ED50/75)
ATO (µM)DCA (mM)
MOLM-14 0.88 ± 0.02 15.9 ± 1.2 0.78 vs 1.60 (2.0) 44 0.803/0.556 
MV4-11 0.53 ± 0.03 19.1 ± 2.3 1.13 vs 2.06 (1.82) 93 
MonoMac6 1.08 ± 0.1 25.1 ± 2.0 0.88 vs 1.9 (2.16) 75 0.807/0.647 
THP-1 4.68 ± 0.13 35.5 ± 0.3 5.37 vs 3.89 (0.72) NA 1.1/0.782 
AML13 0.90 14.0 
AML15 2.6 31.1 
AML16 0.97 0.15 vs 0.97 (6.47) 
IC50IC50 of ATO primed with DCA vs IC50 of ATO alone (PF)Decreased Cell Viability (%)*CI (ED50/75)
ATO (µM)DCA (mM)
MOLM-14 0.88 ± 0.02 15.9 ± 1.2 0.78 vs 1.60 (2.0) 44 0.803/0.556 
MV4-11 0.53 ± 0.03 19.1 ± 2.3 1.13 vs 2.06 (1.82) 93 
MonoMac6 1.08 ± 0.1 25.1 ± 2.0 0.88 vs 1.9 (2.16) 75 0.807/0.647 
THP-1 4.68 ± 0.13 35.5 ± 0.3 5.37 vs 3.89 (0.72) NA 1.1/0.782 
AML13 0.90 14.0 
AML15 2.6 31.1 
AML16 0.97 0.15 vs 0.97 (6.47) 
*

Percentage of increased cell kill when cells sequentially were treated with IC30 of DCA vs vehicle for 48 h and ATO (1 µM) for 48 h. Abbreviations: PF: potentiation factor, ED50/75: effective dose to kill 50%/75% of cells, NA: not applicable.

Conclusion

In AML cells with FLT3 mutations, priming with sublethal dose of DCA, as a mitochondrial enhancer, significantly potentiated the cytotoxicity of ATO. Mechanistic studies on FLT3 signaling in primary AML cells with FLT3-ITD are ongoing. These data suggest that targeting cellular metabolism in leukemia may provide a chemotherapy-free therapeutic option for AML patients who are medically unfit or patients with unfavorable risk such as relapsed/refractory FLT3-ITD and warrants further clinical investigation.

Disclosures:

Off Label Use: Arsenic trioxide in AML.

Author notes

*

Asterisk with author names denotes non-ASH members.