Abstract LB-235: Sensitivity of diffuse large B cell lymphoma cells to Enzastaurin (LY317615.HCl) and its primary metabolite (LY326020) is critically dependent upon disruption of the eIF4F translation initiation complex

Abstract

Abstract Enzastaurin (enza) is currently in phase 3 registration trials for Diffuse Large B Cell Lymphoma (DLBCL) patients at high risk of relapse following R-CHOP therapy. In a phase 2 DLBCL study, 4 of 55 treated patients were progression- free after prolonged, continuous oral enza therapy with 3 of these 4 confirmed as complete responders (Robertson et al., JCO, 2007). The nature of this differential response is unclear, prompting further investigation into the molecular mechanism(s) of enza sensitivity. Enza inhibits PKCβ and related AGC family kinases blocking PKC and PI3K/AKT pathway signaling and inducing apoptosis. In clinical trials, total circulating drug typically reaches steady-state concentrations of 2-4 µM (∼50% enza, ∼50% primary metabolite, LY326020). To identify the critical determinants of sensitivity, we profiled the pro-apoptotic activity of enza and LY326020 in a DLBCL cell panel from both Activated B Cell (ABC) and Germinal Center (GC) subtypes. We show resistant and sensitive DLBCLs from both ABC and GC subtypes. Cells sensitive to enza were also sensitive to LY326020 though, for the first time, we now show that 326020 is decidedly more potent in blocking PI3K-AKT signaling and in inducing apoptosis. In both sensitive and resistant cells, enza and LY326020 reduced phosphorylation of numerous proteins in the PI3K-AKT-TOR pathway (e.g. pGSK3βser9) in a dose and time-dependent manner. However, only sensitive DLBCL cells showed reduced 4EBP1ser65 phosphorylation. By preventing interaction of eIF4E (which binds the 7-mGpppX cap structure of mRNAs) with the scaffolding protein eIF4G, hypophosphorylated 4EBP1 prevents assembly of the translation initiation complex eIF4F (the complex typically elevated in malignancy that preferentially drives translation of potent growth and survival factor mRNAs such as myc, survivin and VEGF). Accordingly, using 7m-GTP co-capture assays to pull down proteins bound to eIF4E, we show a dose and time-dependent increase in 4EBP1: eIF4E binding with concomitant reduction of eIF4E: eIF4G binding. This increase is most pronounced by LY326020. These data indicate that the disruption of the eIF4F complex may be a critical determinant of DLBCL sensitivity to enza and LY326020. Indeed, cells lacking 4EBP1 are insensitive to the pro-apoptotic effects of enza and LY326020, further highlighting the importance of 4EBP1 modulation. Similarly, cells selected for resistance to enza show reduced 4EBP1 expression. These data demonstrate that sensitivity of DLBCL to both enza and LY326020 is critically dependent upon disruption of the eIF4F translation complex. Moreover, these data are the first to show the potent biologic activity of LY326020, the primary metabolite of enza that accounts for ∼50% of total circulating drug in patients and in preclinical models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-235. doi:1538-7445.AM2012-LB-235