Imipramine blue sensitively and selectively targets FLT3-ITD positive acute myeloid leukemia cells

Jonathan Metts,Neil P Shah,Kevin D Bunting,Reuben Kapur,Zhengqi Wang,Jack L Arbiser,Heath L Bradley

doi:10.1038/s41598-017-04796-1

Abstract

Aberrant cytokine signaling initiated from mutant receptor tyrosine kinases (RTKs) provides critical growth and survival signals in high risk acute myeloid leukemia (AML). Inhibitors to FLT3 have already been tested in clinical trials, however, drug resistance limits clinical efficacy. Mutant receptor tyrosine kinases are mislocalized in the endoplasmic reticulum (ER) of AML and play an important role in the non-canonical activation of signal transducer and activator of transcription 5 (STAT5). Here, we have tested a potent new drug called imipramine blue (IB), which is a chimeric molecule with a dual mechanism of action. At 200–300 nM concentrations, IB is a potent inhibitor of STAT5 through liberation of endogenous phosphatase activity following NADPH oxidase (NOX) inhibition. However, at 75–150 nM concentrations, IB was highly effective at killing mutant FLT3-driven AML cells through a similar mechanism as thapsigargin (TG), involving increased cytosolic calcium. IB also potently inhibited survival of primary human FLT3/ITD+ AML cells compared to FLT3/ITDneg cells and spared normal umbilical cord blood cells. Therefore, IB functions through a mechanism involving vulnerability to dysregulated calcium metabolism and the combination of fusing a lipophilic amine to a NOX inhibiting dye shows promise for further pre-clinical development for targeting high risk AML.

Highlights

Acute myeloid leukemia (AML), the most common leukemia in adults and second most common in pediatrics, is a heterogeneous disease with a high relapse rate
Given the prominent role of NADPH Oxidase 4 (NOX4) and reactive oxygen species (ROS) in FLT3/ITD+ acute myeloid leukemia (AML), we sought to determine if FLT3/ITD+ AML might be preferentially sensitive to ROS inhibition
MOLM-14 cells with acquired FLT3 point mutations[17] F691L and D835Y were grown in RPMI + 10% FBS. 32D clone 3 cells transduced with wild-type c-KIT and c-KIT D814V mutation were grown in IMDM + 10% FBS supplemented with 5 ng/mL murine IL-3 (Gemini) and 32D KIT D814V cells were starved of IL-3 for at least 24 hours prior to experimentation

Summary

Introduction

Acute myeloid leukemia (AML), the most common leukemia in adults and second most common in pediatrics, is a heterogeneous disease with a high relapse rate. In FLT3/ITD+ AML, a portion of mislocalized, underglycosylated RTK accumulates on the endoplasmic reticulum (ER) in a highly oxidized microenvironment where the RTK activates signal transducer and activator of transcription 5 (STAT5) through tyrosine phosphorylation, a phenomenon that does not occur with the mature fully glycosylated form, and there are high levels of localized reactive oxygen species (ROS) generated from ER-bound NADPH Oxidase 4 (NOX4)[4, 6]. This non-canonical activation mechanism is leukemia-specific, opening the door to an improved therapeutic index for agents that are able to exploit this STAT5 activation mechanism. We uncovered a new mechanism of action where AML cells are highly sensitive to cytosolic calcium (Ca2+) and vulnerable to Ca2+ overload induced mitochondrial cell death[15, 16]

Methods

Results

Conclusion