ic cells in spleen, liver and bone marrow. Everolimus alone also decreased the percentage of G0 cells in the CD34t leukemic cells of the treated bone marrow. These results indicated the in vivo efficacy of everolimus treatment in a Pht leukemia murine model. Discussion In this study, we showed the effects of everolimus in combination with BMS-582664 VEGFR inhibitor imatinib against Pht ALL quiescent cells. Ex vivo imatinib treatment of Pht leukemia cells from a humanized mouse model showed more residual cells in the CD34tCD38 population, which contains significantly more quiescent cells. Our data showed an ex vivo effect against these residual cells, and the combination of imatinib and everolimus showed an in vivo effect. These data have shown the potential of everolimus to overcome imatinib resistance in quiescent cells.
LSCs are reported to be responsible for the resistance to chemotherapy AMG-208 Flt inhibitor and molecular targeting agents.23,24 In chronic myeloid leukemia, non proliferating quiescent CD34t cells have been found to be more resistant than proliferating leukemic cells after treatment with several chemotherapeutic agents.25 Other studies have shown that inhibitors of mTOR with conventional therapies induced apoptosis and reduced LSCs.8 The definition of LSCs or cancer stem cells is sometimes controversial in certain diseases. In human AML, LSCs have been phenotypically identified within a CD34tCD38 fraction. 23,26 In contrast, it is controversial whether ALL LSCs exist within the CD34t fraction and how CD34, CD38, CD19 and CD133 relate to ALL LSCs.
15,27 29 In our current study, the potential of everolimus to overcome imatinib resistant quiescent cells was demonstrated by using a humanized leukemic mouse model that maintains the differentiation hierarchy of Pht leukemia.15 However, it cannot be determined at this point if the real LSCs of Pht ALL can be diminished until the LSCs in this disease category are clarified. MCL 1, an antiapoptotic member of the BCL 2 protein family, reportedly regulates the self renewal of human hematopoietic stem cells as well as LSCs.30 Mills et al.31 also reported that MCL 1 was translationally regulated by mTORC1. Together with these reports, our results showing decreased expression of MCL 1 by combination treatment of imatinib and everolimus suggested that the combination treatment induced cell death of quiescent Pht leukemia cells by interfering with the mitochondrial mediated cell death pathway.
Rapamycin and its analogs are also known to induce autophagic cell death,32 and Bellodi et al.33 reported that target autophagy potentiates tyrosine kinase induced cell death in Pht leukemia cells. We are also investigating the relation of autophagy in cell death in our experimental systems. In this study, everolimus treatment of Pht leukemia cells from a humanized mouse model decreased the phosphorylation of S6 K, but it increased the phosphorylation of AKT and FOXO1/3a. Rapamycin and its analogs, such as everolimus and temsirolimus, are allosteric mTOR inhibitors that function at a distance from the adenosine triphosphate catalytic binding site. Of the two cellular protein complexes of mTOR molecule, mTORC1 and mTORC2, mTORC1 is sensitive to these allosteric mTOR inhibitors and mTORC2 is resistant.34 mTORC2 directly activates AKT, and this AKT activation in a feedback loop has been reported to correlate with rapamycin failure.35 This feedback