High Throughput Fluorescence-Based In Vitro Experimental Platform for the Identification of Effective Therapies to Overcome Tumour Microenvironment-Mediated Drug Resistance in AML

SIMPLE SUMMARY: The interactions between Acute Myeloid Leukaemia (AML) cells and the surrounding bone marrow (BM) tissue contribute to blocking the efficacy of current drug treatments and to the relapse of patients. Relapsing AML tumours are refractory to current therapies and remain untreatable. Developing new therapies for AML requires the development of new drug-screening methods using in vitro models that closely mimic the interactions of AML cells with cytoprotective BM cells. We have developed a new fluorescence-based in vitro model and an analytical method that takes into consideration the reciprocal interactions between AML cells and the protective BM stroma during drug treatments. Using this new method and combining it with bioinformatics, we have identified new combinations of drugs that may overcome resistance to drug treatments and lead to improved therapies for AML. ABSTRACT: The interactions between Acute Myeloid Leukaemia (AML) leukemic stem cells and the bone marrow (BM) microenvironment play a critical role during AML progression and resistance to drug treatments. Therefore, the identification of novel therapies requires drug-screening methods using in vitro co-culture models that closely recreate the cytoprotective BM setting. We have developed a new fluorescence-based in vitro co-culture system scalable to high throughput for measuring the concomitant effect of drugs on AML cells and the cytoprotective BM microenvironment. eGFP-expressing AML cells are co-cultured in direct contact with mCherry-expressing BM stromal cells for the accurate assessment of proliferation, viability, and signaling in both cell types. This model identified several efficacious compounds that overcome BM stroma-mediated drug resistance against daunorubicin, including the chromosome region maintenance 1 (CRM1/XPO1) inhibitor KPT-330. In silico analysis of genes co-expressed with CRM1, combined with in vitro experiments using our new methodology, also indicates that the combination of KPT-330 with the AURKA pharmacological inhibitor alisertib circumvents the cytoprotection of AML cells mediated by the BM stroma. This new experimental model and analysis provide a more precise screening method for developing improved therapeutics targeting AML cells within the cytoprotective BM microenvironment.