Abstract 1506: Bone marrow microenvironment mediated redox adaptation confers drug resistance in acute lymphoblastic leukemia
Authors: Liu Jizhong , Masurekar Ashish , Holland Mark , Johnson Suzanne , Krishnan Shekhar , Alexander Seema , Parker Catriona , Dempsey Clare , Saha Vaskar 
Published: August 2012

Clinical and laboratory studies in childhood acute lymphoblastic leukemia (ALL) suggest that the bone marrow microenvironment protects blast cells, thus influencing disease relapse. This study was designed to identify the mechanisms by which bone marrow stromal cells could mediate chemoresistance in ALL cells. An in vitro organotypic extracellular matrix-bone marrow stromal cells (ECM-BMSC) culture system, that sustained primary cells without the addition of exogenous factors, was created. ECM-BMSC derived conditioned medium (CM) conferred broad-spectrum chemoprotection to ALL and other cancer cells. Multi drug resistant sub-clones (MDRS), derived by exposing ALL cells in CM to Mitoxantrone, retained chemoresistance even beyond 50 passages in normal medium. Chemoprotective effect was retained in the Proteinase K, RNase and heat resistant, &lt; 3kDa fraction of CM; transmission electron microscopy demonstrated the presence of stromal cell derived exosomes in this fraction. Confocal microscopy confirmed the uptake of ultra-centrifuged fluorescently labelled (PKH 67) stromal exosomes by ALL cells which then developed a resistant phenotype. Stromal derived exosomes and MDRS were enriched in miRNA expression (TaqMan Human miRNA array V3), compared to parent cells, with 92% concordance. Ingenuity™ pathway-based analysis of the unique miRNA pattern predicted dysregulation of mitochondrial function and oxidative phosphorylation. SILAC based phosphoproteomics and U133A V2 gene expression array analysis revealed aberrant PI3K/AKT and oxidative phosphorylation activity. A kinase inhibitor screen showed that PI3K/AKT inhibitors were most potent in reversing CM mediated chemoresistance. Exposure to CM initially (&lt;8 hours) activated pAKT and increased ROS level, partially through stimulating mitochondrial Ca2+ influx. Subsequently (&gt;24 hours), pAKT activity and ROS level decreased to lower than the basal level, with upregulation of CDKN1B and MCL-1, and a decreased mitochondrial membrane potential. shRNA knockdown of CDKN1B restored normal growth rate but not chemosensitivity. MDRS that were resistant to exogenous H2O2 induced apoptosis, regained sensitivity after MCL-1 knockdown. Upregulation of ROS and downregulation of MCL-1 using PEITC lead to cell death in chemo sensitive and resistant cells. Our study identifies that the microenvironment produces soluble factors that induces a redox adaptation in ALL cells leading to chemoresistance. This can be overcome by the simultaneous inhibition of the antioxidant system (to increase ROS) and suppressing antiapoptic proteins (to eliminate the mitochondrial membrane safeguard). Tumour protection by the microenvironment is likely to be common to other cancers. Future therapeutic strategies need to consider targeting the host-tumor interactions in adjunct to conventional chemotherapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1506. doi:1538-7445.AM2012-1506