Biomarkers In Cell Death of Imatinib-Resistant Ph-Leukemia Cells During Treatment with mTOR Inhibitor, Everolimus

Abstract 3988

Aberrant activation of mammalian target of rapamycin (mTOR) signaling pathway has been reported in hematological malignancies including leukemia initiating cells. Although rapamycin and its analogs have proven effective as anticancer agents, the mechanism of action and the solid biomarkers of response have not been fully elucidated. We investigated detailed biomarkers during the cell death of imatinib (IM)-resistant Ph-positive (Ph⁺) leukemia cells due to quiescence or mutations at the ABL-kinase domain after treatment with mTOR inhibitor, everolimus (Eve, RAD001). Ph⁺ leukemic NOD/SCID/IL2rγ^null (NOG) mice cells were long co-cultured with S17 stromal cells and treated with IM and Eve. While slow-cycling (Hoechst 33342^low/Pyronin Y^low) CD34⁺ cells were insensitive to IM in spite of BCR-ABL-dephosphorylation, combination treatment with IM and Eve induced substantial cell death including the CD34⁺ population. In Baf3/p210T315I cells, IM-resistant Ph⁺ leukemia cell line harboring T315I-mutation, Eve also induced cell death with low IC₅₀ values in PI-exclusion assays. In murine model cutaneously injected with Baf3/p210T315I cells, in vivo-treatment with Eve decreased tumor formation. In these systems during treatment with Eve, we did not observe evident dephosphorylations of BCR-ABL, mTOR itself and 4EBP1, but rapid S6K-dephosphorylation with lower doses and decreased expression of MCL-1. Furthermore, the feedback-loop effects such as reversely increased phosphorylations of AKT (Ser473) and FOXO1/3a were also detected during the cell death. We are now investigating more efficient strategies using inhibitors screening kit and also planning to examine new generation of mTOR inhibitors to overcome the IM-resistance due to quiescence or T315I-mutation.