Treatment with the PI3K/mTOR Inhibitor, NVP-BEZ235, Overcomes Resistance to Imatinib in Quiescent or T315I-Mutated Ph⁺ Leukemia Cells,

Abstract 3488

Ph⁺ leukemia is effectively treated with BCR-ABL kinase inhibitors such as imatinib (IM). However, recent studies suggest that IM therapy does not eradicate Ph⁺ leukemia stem cells (LSCs). In order to examine the mechanisms of Ph⁺ LSC resistance to IM and seek strategies to overcome this, Ph⁺ ALL patient cells were serially xenotransplanted into immunodeficient NOD/SCID/IL2rγ^null (NOG) mice and abundant spleen cells derived from leukemic NOG mice were co-cultured with S17-stromal cells using methods previously reported (Minami et al., Proc Natl Acad Sci USA, 2008). Slow-cycling (Hoechst-33342^low/Pyronin-Y^low) CD34⁺ cells were insensitive to IM despite complete dephosphorylations of BCR-ABL and its substrate CrkL, which indicated that addiction of BCR-ABL activity is lower for survival in such quiescent cells. From comprehensive drug screening of other small compounds using this co-culturing system, we found that inhibitors of PI3K/AKT/mTOR-axis signaling, including rapamycin, were promising candidates. Aberrant activation of mTOR signaling has also been reported to be involved in some systems of leukemia initiating cells. In vitro, combination treatment with IM and rapamycin analogue, everolimus (RAD001), induced substantial cell death in the slow-cycling CD34⁺ population with p70-S6K dephosphorylation and decreased expression of the anti-apoptotic BCL-2 family protein, MCL-1. In the leukemic NOD/SCID mouse ALL model, in vivo RAD001-treatment showed a decrease of tumor burden including slow-cycling CD34⁺ cells. However, during the cell death by RAD001-treatment, however, negative feedback-loop effects were detected such as reversely increased phosphorylations of AKT (Ser473) and FOXO1/3a. In vitro, the dual PI3K/mTOR inhibitor, NVP-BEZ235 (BEZ), induced substantial cell death including slow-cycling CD34⁺ cells at lower doses than those required by RAD001. In biomarker analyses, BEZ-treatment inhibited not only p70-S6K phosphorylation but also phosphorylations of mTOR (Ser2481) itself, 4E-BP1, AKT (Ser473) and FOXO1/3a. At the same time, expression of MCL-1 decreased during the cell death by BEZ-treatment. In T315I-mutated BCR-ABL dependent cell lines such as murine Baf3/p210-T315I and human-derived TCC-Y/T315I, treatment with BEZ induced growth inhibition and cell death. The effects of BEZ on normal stem cells and in mouse Ph⁺ leukemia models are under investigation. These results suggest that treatment with BEZ might have potential for overcoming the resistance to IM due to quiescent property in Ph⁺ LSCs or T315I-mutation.