Mechanism Of Anti-Myeloma Effects Due To Silencing Of Deptor

DEPTOR is a recently described 48 kDa protein that binds to mTOR and inhibits this kinase within TORC1 and TORC2 complexes. Over-expression of DEPTOR specifically occurs in the multiple myeloma genetic sub-class that contains IgH translocations. Most interestingly, DEPTOR knock-down in over-expressing MM cell lines induces growth arrest and apoptosis, indicating DEPTOR could serve as a therapeutic target in selected patients. To investigate the mechanism of MM cytoreduction induced by DEPTOR silencing we generated a doxycycline-inducible DEPTOR shRNA. Induced DEPTOR knockdown in 8226 and OPM-2 cell lines resulted in phosphorylation of p70S6K and 4E-BP1 (due to release of TORC1 inhibition) as well as apoptosis, cell cycle arrest and senescence. RAPTOR knockdown in these MM cells prevented TORC1 stimulation and the anti-MM effects resulting from DEPTOR silencing. Thus, the acute activation of TORC1 mediated the apoptosis and growth arrest. Although DEPTOR silencing and mTORC1 activation resulted in depressed AKT activity, probably due to the TORC1-PI3K/AKT negative feedback loop, this was not relevant to anti-MM effects as over-expression of wild type or a phosphomimetic version of AKT did not prevent the adverse effects. Similarly, induction of ER stress was also not relevant to the anti-MM effects as markers for UPR activation did not occur during DEPTOR silencing. Although western blot did not show alteration of p53 expression, p21 and p27 were induced subsequent to DEPTOR knockdown. Associated RAPTOR knockdown prevented the upregulation of p21 but did not affect p27 levels. In addition, over-expression of AKT did not prevent the upregulation of p21. To test whether p21 was integral to DEPTOR knockdown effects, we silenced p21. P21-silenced MM cells were protected against apoptosis, cell cycle arrest and senescence induced by DEPTOR knockdown. Furthermore, DEPTOR knockdown in a murine xenograft model, achieved by ingestion of doxycyline, resulted in tumor regression, TORC1 activation, apoptosis and p21 induction. These data indicate a novel pathway to explain the lethal effect of DEPTOR knockdown in MM cells. The pathway is initiated by acute activation of mTORC1, with subsequent p21 induction. The data also suggest that DEPTOR over-expression in some MM clones is pro-tumoral due to its ability to suppress expression of CDK inhibitors like p21 and p27.