Determining the mechanism behind imipramine blue’s selectivity towards FLT3-ITD+ acute myeloid leukemia Open Access

Abstract

FLT3-ITD⁺ acute myeloid leukemia (AML) accounts for a quarter of AML cases and is associated with very poor prognosis and high relapse rate. Although in recent years many FLT3 inhibitors have been extensively studied, they only produced transient responses. Imipramine blue (IB) and honokiol (HK) has emerged as promising new drugs to treat FLT3-ITD⁺ AML targeting oncogenic reactive oxygen species (ROS) and signal transducer and activator of transcription 5 (STAT5). IB is found to cause cytosolic calcium release and more potent at inducing cell death in FLT3-ITD⁺ AML cell lines than FLT3-WT. This thesis primarily aims to figure out why IB exhibits greater efficacy towards FLT3-ITD⁺ AML. Contrary to our hypothesis, we found that IB did not induce ER stress in AML cell lines. We also discovered that dynamin related protein1 (Drp1), a key component of mitochondrial fission, had higher expression in FLT3-ITD⁺ cell lines and IB treatment activated Drp1. Therefore, we proposed that more Drp1 had been activated by IB in FLT3-ITD⁺ AML, leading to extensive mitochondrial fission and subsequently more robust apoptosis. However, inhibiting IB-induced activation of Drp1 did not eliminate the difference in sensitivity towards IB between FLT3-ITD⁺ cell lines and FLT3-WT cell line. On the other hand, HK formed potent synergy with pimozide, an FDA-approved anti-psychotic drug.

Table of Contents

Table of Contents

Introduction

1

Acute myeloid leukemia (AML) overview

2

FLT3-ITD⁺ AML

      

2-3

          Positive feedback loop between STAT5 and ROS

3-4

          IB as potential therapy targeting FLT3-ITD⁺ AML

4-5

          Mitochondria-ER crosstalk

6-7

          ER stress and unfolded protein response (UPR)

8

Dynamin related protein (Drp1) in cancer

9

Honokiol (HK) as an alternative

9-10

Scope of the Thesis

11

Materials and Methods

12

Cell culture

13

Treatment reagents

13

Trypan blue exclusion assay

13

Mitochondrial mass measurement

13

RT-PCR                                                                                                          

13-14

XBP1 splicing assay

14

Western blotting

14

Data analysis

       15

Results

16

IB exhibited greater potency in FLT3-ITD⁺ cell lines

17

Drp1 mRNA expression was higher in FLT3-ITD⁺ cell lines

18-19

Drp1 protein expression was higher in FLT3-ITD⁺ cell lines and

19-21

IB increased Drp1 activation

Calcineurin inhibition did not ablate the difference in IB

22-23

sensitivity between FLT3-ITD⁺ and FLT3-WT cell lines

Unlike thapsigargin (TG), IB did not induce ER stress in AML cell

24-25

lines

Honokiol synergized with pimozide

26-27

Discussion

28-30

References

31-32

Appendix I: primer list

33-34

Appendix II: Supplementary Figure

35-36

About this Master's Thesis

Rights statement

• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	M.S.
Submission	Master's Thesis
Language	English
Research Field	Biology, Cell Health Sciences, Oncology
Keyword	FLT3-ITD acute myeloid leukemia imipramine blue
Committee Chair / Thesis Advisor	Kevin D. Bunting, Emory University
Committee Members	Cheng-Kui Qu, Emory University Lily Yang, Emory University

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