Inhibition Of The MTORC2and Chaperon Pathways To Treat Leukemia

Constitutive activation of the kinases AKT or PKC in blood cancers promotes tumor cell proliferation and survival and is associated with poor patient survival. The mammalian target of rapamycin (mTOR) complex2(mTORC2) regulates the stability of AKT and classical (c)PKC (PKCa&PKCβ) proteins by phosphorylating the highly conserved turn motif (TM) of these proteins. In cells that lack mTORC2function, the TM phosphorylation of AKT and cPKC is abolished. Consequently, AKT and cPKC protein stability is impaired, however the chaperone protein HSP90can rescue AKT and cPKC stability and restore the expression of these proteins. In this study we investigate the anti-tumor effects of inhibiting mTORC2plus HSP90in mouse and human leukemia cell models. We show that the HSP90inhibitor17-AAG preferentially inhibits AKT expression and promotes cell death in mTORC2deficient pre-B leukemia cells. Furthermore, we show that17-AAG selectively inhibits mTORC2deficient leukemia cell growth in vivo. Finally, we show that treating human leukemia cells with the mTOR inhibitor rapamycin plus17-AAG, but not rapamycin or17-AAG alone, inhibits AKT expression and preferentially promotes leukemia cell death. These studies provide a mechanistic and clinical rationale to combine mTOR inhibitors with chaperone protein inhibitors to treat human blood cancers.There are three parts in our study.Part I Inhibition of the mTORC2and chaperon pathways to destabilize AKTObjective Inhibiton of the mTORC2and chaperon pathways will destabilize AKT in the leukemia cell of both mouse and human. The study will us the theoretical principle to combine mTORC2and chaperon pathways inhibitor to treat leukemia.Methods①uilding our cell lines with transfecting pro-B cells with Ab-MuLV or p210BCR-Abl virals;②use the gene sequence of human sinl to reconstitute sinl-/-leukemia cells to restore the mTORC2functions;③treat cell lines with17AAG or17AAg+rapamycin for certain time, then run Western Blot to measure AKT protein level.Results①AKT will destabilize in sinl-/-leukemia cells after17AAG treatment for just4hours;②restore mTORC2function will protect AKT from destabilizing after17AAG treatment;③treat wild type leukemia cellsw with rapamycin and17AAG will destabilize AKT.Conclusion Inhibiton of the mTORC2and chaperon pathways will destabilize AKT in the leukemia cell Part2Inhibition of the mTORC2and chaperon pathways will induce leukemia cell death in vitroObjective To test whether the mTORC2and HSP90inhibitors will induce leukemia cell death in vitroMethod treat cell lines with17AAG or17AAg+rapamycin for certain time,①then incubated with Propidium Iodide (PI) and Annexin V-PE in Annexin V binding buffer (10mM HEPES, pH7.3,150mM NaCl,1.8mM CaCl2) at room temperature for15minutes then analyzed by flow cytometry.②live cells were counted by trypan blue cell viability assay. Results①17AAG preferentially induce cell death in sinl-/-leuk-emia cells;②Restoration of mTORC2function protects sinl-/-leukemia cells from17AAG mediated cell death;③Co-administration of rapa-mycin and17AAG promotes leukemia cell death.Conclusion Inhibition of the mTORC2and chaperon pathways will induce leukemia cell death in vitro. Part3Inhibition of the mTORC2and chaperon pathways will induce leukemia cell death in vivoObjective To prove inhibiton both mTORC2and HSP90functions will induce leukemia cell death in vivo.Method BCR-Abl-transformed pre-B cells were harvested in culture and injected via tail vein into sub-lethally irradiated (300cGy) syngeneic Peb3b (CD45.1+) mice.17-AAG was administered by one intraperitoneal injection immediately following cell transfer, and repeated daily for5days. Mice were killed at day7(day6was not treated) to harvest bone marrow and spleen. Leukemic engraftment was determined by flow cytometry.Results①17-AAG treatment resulted in a significant decrease in the proportion and total number of Sin1-/-pre-B ALL cells in the bone marrow while17-AAG treatment caused no significant decrease in the number of Sin1+/+leukemia cells.②17-AAG treatment resulted in a significant decrease in the proportion and total number of Sin l-/-pre-B ALL cells in the spleen while17-AAG treatment caused no significant decrease in the number of Sinl+/+leukemia cells Conclusion Inhibition of the mTORC2and chaperon pathways will induce leukemia cell death in vivo...