Dual Inhibition of mTORC1/mTORC2 Induces Apoptosis of Mantle Cell Lymphoma by Preventing Rictor Mediated AKT^S473 Phosphorylation by Potentiating AKT2-PHLPP1 Association

Abstract 772

The mammalian target of rapamycin (mTOR) plays a crucial role in proliferative and anti-apoptotic signals in various lymphomas. The mTORC1 inhibitors, such as the rapamycin analogs temsirolimus and everolimus, have achieved a 38% overall response rate in heavily pretreated mantle cell lymphoma (MCL); however, most responses are partial, and many patients are resistant. One mechanism for resistance to mTORC1 inhibitors is that mTORC2 remains unaffected (Blood 2009 Oct 1; 114 (14): 2926–2935). We hypothesize that combined mTORC1/mTORC2 inhibition will be more effective in MCL. We tested the dual mTORC1/mTORC2 inhibitor OSI-027 (OSI Pharmaceuticals) on MCL cell lines and patient samples that were demonstrated to have a high amount of mTORC2 (Rictor and its target p-AKT^S473) and mTORC1 (Raptor and its targets pS6 and p4E-BP1) targets. Dual inhibition through OSI-027 inhibited pAKT^Ser473, a direct target of mTORC2 without any obvious effect on AKT^Th308 in these cells. On the other hand, the mTORC1 inhibitor rapamycin had no effect on pAKT^Ser473. Interestingly, both drugs effectively suppressed phosphorylation of S6; only OSI-027 decreased phosphorylation of 4E-BP1^Th37/46. We next assessed the effect of OSI-027 on MCL proliferation and apoptosis and showed that dual inhibition of mTORC1/mTORC2 through OSI-027 caused growth arrest and apoptosis in a dose-dependent manner in both MCL patient cells treated ex vivo and established MCL cell lines; however, we did not see any significant effect of rapamycin on apoptosis. Recent studies have shown that AKT has direct effects on some members of the apoptosis pathway such as forkhead transcription factors (FOXO), caspase 9, and BAD. OSI-027 treatment significantly inhibited p-FOXO3A^T32 with some inhibitory effect on pBAD^S136. These data suggest that FOXO3A is an important target of AKT through which OSI-027 is exerting its apoptotic effects. There are several potential mechanisms whereby OSI-027 prevents AKT^S473 phosphorylation: 1) deactivation of kinases upstream of AKT; 2) activation of phosphatases down-stream of AKT. OSI-027 treatment of MCL cells produced no change in phosphorylated PI3K p85/p55 or PDK1 protein. These data eliminate the possibility of involvement of the PI3K pathway in OSI-027 mediated AKT dephosphorylation. We did find that OSI-027 mediated deactivation of AKT^S473 is, at least in part, phosphatase dependent. The PH domain leucine-rich repeat protein phosphatase (PHLPP) family of phosphatases have been shown to directly dephosphorylate and inactivate AKT^S473. To determine if AKT and PHLPP1 or PHLPP2 are associated in MCL cells, we immunoprecipitated AKT from MCL cells and probed for association with endogenous PHLPP1 and PHLPP2. All the MCL lines do express endogenous PHLPP1 and PHLPP2; however, they are not complexed to AKT. Further studies using the AKT isoforms AKT1 and AKT2 found AKT2 to be associated with PHLPP1; there was no association of AKT1 or AKT2 to PHLPP2. To investigate if OSI-027 enhanced the AKT2-PHLPP1 interaction, we repeated the co-immunoprecipitation assay in the OSI-027 treated MCL cell lines and demonstrated that OSI-027 increased the interaction of AKT2-PHLPP1. Further we showed that OSI-027 exposure did not alter the expression level of PHLPP1 and PHLPP2 proteins. In summary the dual inhibition of mTORC1/mTORC2 by OSI-027 in MCL is more effective than mTORC1 inhibition by rapamycin. We present the mechanism that OSI-027 induces apoptosis in MCL cell lines by inhibition of Rictor-AKT-FOXO3A signaling, and this is mediated in part by activation of the phosphatase PHLPP1. These findings indicate that simultaneously targeting mTORC1 and mTORC2 may be effective in patients with MCL.