Mitochondrial Profiling of Acute Myeloid Leukemia in the Assessment of Response to Apoptosis Modulating Drugs.

Jo Ishizawa,Michael Andreeff,E J Dettman,Sharon Shacham,Dhruv Chachad,Kensuke Kojima,Graciela M Nogueras-Gonzalez,Michael H Cardone,Xuelin Huang,Teresa Mcqueen,Marina Konopleva,Vivian Ruvolo,William E Pierceall,Dhyan Chandra

doi:10.1371/journal.pone.0138377

Abstract

BH3 profiling measures the propensity of transformed cells to undergo intrinsic apoptosis and is determined by exposing cells to BH3-mimicking peptides. We hypothesized that basal levels of prosurvival BCL-2 family proteins may modulate the predictive power of BH3 profiling and termed it mitochondrial profiling. We investigated the correlation between cell sensitivity to apoptogenic agents and mitochondrial profiling, using a panel of acute myeloid leukemias induced to undergo apoptosis by exposure to cytarabine, the BH3 mimetic ABT-199, the MDM2 inhibitor Nutlin-3a, or the CRM1 inhibitor KPT-330. We found that the apoptogenic efficacies of ABT-199 and cytarabine correlated well with BH3 profiling reflecting BCL2, but not BCL-XL or MCL-1 dependence. Baseline BCL-2 protein expression analysis increased the ability of BH3 profiling to predict resistance mediated by MCL-1. By utilizing engineered cells with overexpression or knockdown of BCL-2 family proteins, Ara-C was found to be independent, while ABT-199 was dependent on BCL-XL. BCL-2 and BCL-XL overexpression mediated resistance to KPT-330 which was not reflected in the BH3 profiling assay, or in baseline BCL-2 protein levels. In conclusion, mitochondrial profiling, the combination of BH3 profiling and prosurvival BCL-2 family protein analysis, represents an improved approach to predict efficacy of diverse agents in AML and may have utility in the design of more effective drug combinations.

Highlights

Treatment outcomes for acute myeloid leukemia (AML) are generally better than those achievable for many other malignancies
We investigated the correlations among factors related to cell death and BCL-2 family proteins in AML by: 1) analysis of apoptosis of AML cells by four different anti-leukemia compounds: cytarabine (Ara-C), the BH3-mimetic ABT-199, the MDM2-inhibitor Nutlin-3a, and the XPO1-inhibitor KPT-330; 2) BH3 profiling of the AML cells; and 3) determination of basal protein expression levels of BCL-2, MCL-1, and BCL-XL (Figure A in S1 File)
We conclude that mitochondrial profiling, by incorporating classical quantification of basal expression of BCL-2 family proteins with BH3 profiling, can characterize the dependence on BCL-2 family proteins of agent-specific apoptosis more precisely than could be done by BH3 profiling alone

Summary

Introduction

Treatment outcomes for acute myeloid leukemia (AML) are generally better than those achievable for many other malignancies. Incorporating quantitation of basal expression levels of BCL-2 family proteins measured by classical methods (e.g., immunoblotting) may provide additional information for the assessment of sensitivity or resistance of cells to chemotherapeutic agents. We investigated the correlations among factors related to cell death and BCL-2 family proteins in AML by: 1) analysis of apoptosis of AML cells by four different anti-leukemia compounds: cytarabine (Ara-C), the BH3-mimetic ABT-199, the MDM2-inhibitor Nutlin-3a, and the XPO1-inhibitor KPT-330; 2) BH3 profiling of the AML cells; and 3) determination of basal protein expression levels of BCL-2, MCL-1, and BCL-XL (Figure A in S1 File). Our study demonstrates that mitochondrial profiling, a combined assessment of BH3 profiling and the basal expression of BCL-2 family proteins, is a potentially improved approach to predicting activity of antitumor agents

Materials and Methods

Results

Discussion