Abstract B06: Overcoming hypoxia-induced resistance is essential for effective survivin targeting in acute myeloid leukemia
Authors: Lee David W. , Montoya Justin , Finch Kieran , Wilkinson-Dix Jack , Arceci Robert J. , Wai Daniel H. , Aleem Eiman 
Published: February 2019

Abstract
Background: Although the clinical outcome of pediatric acute myeloid leukemia (AML) has improved over the past few decades, approximately 30% of patients relapse. The overall survival (OS) rate of pediatric patients with primary refractory or relapsed AML is about 30%. Resistance to therapy presents a great challenge for AML patients. Therefore, there is a critical need to understand mechanisms of resistance, as well as to identify novel therapies that will improve OS. The leukemia-initiating cells reside in special hypoxic niches in the bone marrow. Thus, studying drug response of AML cells within a hypoxic environment is essential for more accurate prediction of patients’ response to therapy. We performed functional drug screening using AML cell lines, which demonstrated a differential response of AML cell lines to the survivin-targeting agent YM155 in normoxia versus hypoxia. Survivin (BIRC5), an inhibitor of apoptosis, is overexpressed in a variety of pediatric blood cancers compared to normal cells. We hypothesize that survivin may be an attractive target for therapy in pediatric AML. The overall objective of this study was to study mechanisms of AML response to YM155 in normoxia and hypoxia and survivin’s potential as a therapeutic target in pediatric AML.
Purpose: The aims of the present work were (1) to study BIRC5 expression in publicly available databases of pediatric AML including the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative, and (2) to determine the potential mechanisms of hypoxia-altered response to YM155 in AML cells.
Methods and Results: In the present study, the TARGET AML gene-expression profiling data available on Affymetrix human gene 1.1 ST arrays (HuGene 1.1) were used to study BIRC5 expression in comparison to HuGene 1.1 datasets from Gene Expression Omnibus (GEO) containing profiling data from adult AML and normal tissues. BIRC5 expression was significantly higher in pediatric AML in comparison to cytogenetically normal (CN) adult AML, and in relapsed pediatric AML in comparison to matched samples at diagnosis. In order to identify novel targeted therapies in AML we used a panel of AML cell lines to screen a library of 50 compounds in normoxia and hypoxia. The survivin-targeting agent YM155 was found to be one of the most cytotoxic compounds in the majority of AML cell lines in normoxia. However, hypoxia conferred resistance to this drug. The IC50 of YM155 in AML cell lines in normoxia ranged from 1.5 nM-8 µM, with an increase of 2-100 fold in hypoxia (IC50 from 22 nM-14 µM). The Mv4-11 cell line that harbors a FLT3-ITD mutation was selected for further mechanistic studies. Hypoxia conferred resistance to YM155 in Mv4-11 cells through inhibition of apoptosis. In order to identify the underlying molecular mechanisms, we performed RNA-seq of YM155-treated Mv4-11 cells in normoxia and hypoxia, and identified a set of over-represented genes that were validated by quantitative RT-PCR. The mRNA expression of HIF2alpha, p21WAF1, and STAT3 was overexpressed after 9h of YM155 treatment, while PIM2 was downregulated in normoxia but not in hypoxia, in comparison to vehicle-treated control. Preliminary data suggest a role of stem cell genes in hypoxia-induced resistance to YM155. Ongoing studies are directed towards determining the functions of the identified genes in resistance.
Conclusion: Our data demonstrate that survivin (BIRC5) is overexpressed in pediatric AML in comparison to adult CN-AML, and may be an attractive target for pediatric AML therapy. Additionally, YM155 is highly cytotoxic to AML cell lines in normoxia; however, for its effective use in the clinic, cotargeting hypoxia-induced resistance factors is essential.
Citation Format: David W. Lee, Justin Montoya, Kieran Finch, Jack Wilkinson-Dix, Robert J. Arceci, Daniel H. Wai, Eiman Aleem. Overcoming hypoxia-induced resistance is essential for effective survivin targeting in acute myeloid leukemia [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B06.