Enzastaurin Displays Activity against Follicular Lymphoma Cells: Role of mTOR Pathway

Recent preclinical and clinical studies using the macrocyclic bisindolylmaleimide enzastaurin or the N-benzylstaurosporine midostaurin demonstrate promising activity of PKC inhibitors in a variety of tumors, including diffuse large B-cell lymphoma, multiple myeloma and Waldenström’s macroglobulinemia. However, the potential of these drugs have not been investigated in follicular lymphoma (FL). In this study, we found that enzastaurin inhibited cell proliferation of FL RL and DOHH-2 cells as measured by a viability assay (IC50 = 3–5 μM. In our hands, RL cells displayed similar sensitivity to enzastaurin than diffuse large B lymphoma cells (DLBCL). Enzastaurin induced apoptosis in a dose-dependent manner (40% apoptotic cells at 5μM measured by DAPI staining). Based on our previous studies demonstrating the critical role of mTOR pathway in FL cell proliferation and survival (Leseux et al., Blood 2006), we investigated the effect of enzastaurin on mTOR regulators and targets. Here, we show for the first time that enzastaurin inhibited mTOR-dependent phosphorylation of p70-S6 kinase (Thr389), S6 ribosomal protein, and 4EBP1, all proteins being directly or indirectly targeted by mTOR. The magnitude of mTOR inhibition was found to be comparable to that observed with rapamycin. Moreover, in RL cells, enzastaurin had no impact on Syk, Akt, and ERK phosphorylation, all enzymes being involved in mTOR regulation in these cells. However, we found that enzastaurin also induced a dramatic dose-dependent accumulation of beta-catenin followed its partial degradation. This observation conflicts with total inhibition of GSK3-beta phosphorylation at Ser9 site, resulting in GSK3-beta activation. K. Anderson’s group (ASH meeting 2007, Abstract # 258) had already described this paradoxical finding in multiple myeloma cells. Additional studies showed that, in these cells, enzastaurin had a modest inhibitory effect on global PKC activity and influenced neither Bcl2 phosphorylation profile at Ser70, nor Bcl2 expression level. We were unable to identify higher anti-leukemic effect of Enzastaurin and Rituximab combination, compared to enzastaurin or Rituximab alone, as previously described in Waldenström’s disease cellular models. The lack of cooperation between the two molecules in FL cells was not surprising since we have published that mTOR is also a target for Rituximab (Leseux et al., Blood 2008). Altogether, these results suggest that enzastaurin exerts a potent pro-apoptotic effect in FL cells in spite of high Bcl2 expression. Whereas the role of beta-catenin pathway deserves further clarification for enzastaurin mechanism of action, this study provides evidence that mTOR inhibition plays an important role, suggesting that mTOR status could be an important parameter to select patients for enzastaurin therapy. Finally, this study supports clinical studies based on the use of enzastaurin as maintenance therapy in FL as it is currently used in the ongoing international PRELUDE study for DLBCL patients.