A laboratory assay for improved prediction of drug responses in acute myeloid leukaemia cells

Abstract

Abstract Background The five-year-survival-rate for paediatric patients with Acute Myeloid Leukaemia (AML) is from 60-70%. The most common causes of death are disease relapse and chemo-resistance. Typically, drug dose response testing is performed at atmospheric oxygen (21%) in a 2-Dimensional (2-D) format; however, AML cells reside in the hypoxic (1% O2), 3-Dimensional (3-D) bone marrow structure along with other cells. Therefore, the drug response observed in typical culture conditions (2D, 21% O2) do not always translate to the same response in patients. The objectives of the present study were to: (1) develop an in vitro assay reproducing key elements of the bone marrow microenvironment (BMM), and to (2) use this system to evaluate the sensitivity/resistance of AML cell lines towards selected therapeutic agents. Methods A functional drug screen was performed first in 2D normoxia and hypoxia to evaluate the response of AML cell lines to 50 compounds. RNA sequencing was performed to determine global changes of gene expression. Overrepresented genes were further matched to potential pathways using Ingenuity Pathway Analysis. Results were validated using real-time RT-PCR. MV-4-11, an AML cell line with a FLT3 mutation was used to study the effects of 3-D structure on drug response. Cells were seeded in 3D and allowed to grow for 72h, compounds were added for an additional 24 h and cell viability was measured using Cell Titer Glo assay. Results Twelve cell lines were used in the present study. In the initial screen THP1 cells for example were more sensitive in hypoxia to 8 out of 50 different compounds. In MV-4-11 cells, hypoxia and 3-D structure conferred resistance towards YM155, a BIRC5 targeting compound. Mechanistic studies revealed that MCL-1 stabilization may underlie this response. A functional drug screen using 50 compounds in 3-D showed that 10 out of 50 compounds resulted in significantly reduced viability of MV-4-11 cells compared to vehicle indicating that this assay can be used for high-throughput drug screening (HTS). Conclusions Hypoxia and 3D structure alter drug response in AML cells. The proposed 3D assay can be used for HTS. Clinical application of this study may lead to more accurate predictions of therapeutic outcomes. Legal entity responsible for the study The authors. Funding Children Cancer Network. Disclosure All authors have declared no conflicts of interest.