Histone Deacetylase Inhibition with LBH589 Inhibits the Rapamycin Insensitive Rictor-mTOR (mTORC2) Complex and Translation Initiation Factor eIF4E Activation in Diffuse Large B-Cell Lymphoma

Diffuse large B cell lymphoma (DLBCL) is an aggressive form of non-Hodgkin lymphoma and the treatment of these patients has improved with rituximab-based chemo-immunotherapy. However, approximately 40% of patients die of their disease and new agents with novel mechanisms of action are needed for this disease. The mammalian target of rapamycin (mTOR) has emerged as an important therapeutic target in cancer cells and rapamycin and its derivatives, which specifically inhibit mTOR, are now being actively evaluated. Recent clinical trials of mTOR inhibitors have demonstrated that a fraction of patients (35%) with relapsed DLBCL respond to single-agent temsirolimus (Smith S et al J Clin Oncol 26; May 20 Supplement abstract 8514) or everolimus (Reeder et al 2007 ASH Annual Meeting Abstracts 110 (11) abstract 121) in relapsed DLBCL, however most patients are resistant to this therapy. The mechanism of this resistance remains a subject of major therapeutic significance. Herein, we report that cells from DLBCL lines (DHL-6, Ly7 and Ly3) and primary tissues from DLBCL patients (n=10) display persistent activation of the mTOR pathway as determined by phosphorylation of mTOR targets S6 ribosomal protein (S6rp) and 4E-binding protein 1 (4E-BP1). Treatment of human DLBCL cells with various doses of rapamycin predictably demonstrated a decrease in proliferation but less than 10% reduction in overall cell survival. However, rapamycin suppressed the phosphorylation of S6rp and 4E-BP1, indicating an inhibition of raptor-mTOR (mTORC1) signaling. Paradoxically, rapamycin also concurrently increased, through a negative feedback mechanism, the phosphorylation of Akt that may contribute to drug resistance. Interestingly we found that rapamycin treatment also increased the phosphorylation of eIF4E, a survival protein downstream of mTOR, which may also be responsible for resistance of rapamycin along with Akt. To determine whether the observations found in vitro are clinically relevant, we obtained peripheral blood samples from patients with aggressive lymphoma treated with the rapamycin derivative everolimus. The levels of phosphorylated Akt and eIF4E were increased in 3 of 3 patient samples at 48 hrs and after 1 cycle compared to untreated control. Our observations therefore suggest that rapamycin derivatives potently activate Akt and eIF4E activity via activation of the mTORC2 assembly, in addition to its well-characterized ability to suppress the mTORC1 pathway. Treatment of DLBCL cells with histone deacetylase (HDAC) inhibitor LBH589 induced growth inhibition of DLBCL cells at nanomolar concentrations in a dose dependent manner in association with hyperacetylation of histones H3 and H4. LBH alone was able to inhibit the phosphorylation of S6rp and 4E-BP1, while combined treatment with rapamycin inhibited the phosphorylation of S6rp and 4EBP1 to a greater extent than either agent alone. Surprisingly LBH inhibited constitutive as well as rapamycin-induced activation of Akt and eIF4E in a time and dose dependent manner. Our co-immunoprecipitation data suggest that LBH alone was able to alter the level of intact mTORC2 by reducing the amounts of rictor bound to mTOR, which is further decreased when combined with rapamycin. In support of this model, rapamycin combined with LBH exhibited enhanced synergistic inhibitory effects on survival and proliferation of DLBCL cells. The mTOR pathway is also considered to control the translation of specific mRNA species, some of which are involved in cell cycle control and angiogenesis (e.g. cyclin D1, c-Myc and HIF-1a). LBH alone down-regulated expression of c-Myc and HIF-1a, while have no effect on cyclin D1 expression. Combination of LBH with rapamycin further decreased expression of c-Myc and HIF-1a. Overall these results indicate that pharmacological inhibition of the mTOR pathway by rapamycin and LBH interferes with essential survival and proliferating pathways in DLBCL cells. We propose that the rapamycin-induced functional blockade of Akt and eIF4E is inhibited by LBH, and the combination increased the anti tumor activity of rapamycin. In summary, our data provide a mechanistic basis for enhancing mTORtargeted cancer therapy by combining an mTOR inhibitor with a HDAC inhibitor such as LBH. A phase I/II trial of this combination is planned.