Inhibitors Of Farnesyltransferase and Everolimus Act Synergistically In Growth Inhibition Of Human T-NHL Cells By Involvement Of AMPK
Authors: Melchinger Wolfgang , Zierock Leonidas , Wehrle Bettina , Marks Reinhard 
Published: October 2019

Abstract

Introduction
The outcome of patients with T cell lymphoma treated with standard chemoterapeutic substances remain poor, making the search for new active substances a highly medical need in this hematologic neoplasia. Recent phase II clinical trials showed very promising activity of farnesyltransferaseinhibitors (FTI) in relapsed/refractory T-NHL patients (Witzig et al. 2011). Regarding the molecular mechanisms behind this therapeutic effect, conflicting data regarding Ras as the initially proposed intracellular target of FTI and the involvement of MAP kinases in cellular effects of FTI in T cells exist (Marks et al. 2007, Ding et al. 2011). Together with observations in breast and ovarian cancer cells suggesting the GTPase Rheb as target for inhibition of farnesylation (Basso et al. 2005), the targets of FTI might vary according to the examined cell type. Interestingly, in breast cancer cells FTI mediatied inhibition of Rheb action resulted in reduced mTOR signaling. Nevertheless, as a putative additional targeted treatment approach in T-NHL, incubation with mTOR inhibitors showed not only substantial antiproliferative effects in normal T cells but also in malignant human T cell lymphoma lines in vitro (Huang et al. 2010). Since further clinical trials with both substances did not show severe side effects, adding everolimus as combination partner might even enhance clinical activity of FTI in T cell lymphomas. Therefore, in order to test this hypothesis and to analyse if both substances differ in their molecular mechanisms of action, FTI and everolimus were tested in vitro in T cell lymphoma lines (Karpas, Derl-2, Jurkat) to evaluate potential synergistic modes of action.

Methods and Results
Incubation of human T cell lymphoma lines Karpas and Derl-2 with the FTI SCH66336 (lonafarnib) or the mTOR inhibitor everolimus showed a reduction in proliferation in a dose dependent manner (EC50 for everolimus: 0.1nM, EC50 for lonafarnib: 0.5 µM). Combining both drugs resulted in synergistic inhibition of proliferation. This inhibitory effect correlated with increased p27KIP1 expression. In our experiments, Rheb appeared to be highly expressed in all examined T cell lymphoma lines with even additional increase of protein expression in Karpas cells after FTI incubation. Comparing FTI action to inhibition of mTOR by everolimus on a molecular level, in our experiments lonafarnib treatment of Karpas cells resulted in an unexpected reduction in AMPK-phosphorylation, implicating involvement of this metabolic pathway in FTI mediated inhibition of proliferation in malignant T cells. This effect could not be observed in everolimus treated Karpas cells. In contrast, naive human CD4+ T cells showed very little Rheb protein expression, which could be significantly increased after TCR stimulation by induction of Rheb mRNA transcription. While everolimus treatment of TCR-activated normal human CD4+ T cells resulted in AKT-hyperphosphorylation, FTI did not induce any changes in AKT. Contrary to the malignant T cells, FTI treatment had no impact of AMPK phosphorylation in activated T cells. Actually, naive T cells treated with FTI showed an hyperphosphorylated AMPK status.

Conclusion
Lonafarnib and everolimus show synergistic antiproliferative effects in T cell lymphoma lines, most likely by interfering with mTOR and AMPK signalling, making this combination therapy interesting for clinical trials. In contrast, FTI does not mediate AMPK in activated normal T cells. This observations are in accordance with a differential targeting of Rheb by FTI in malignant or normal human T cells.

Disclosures:
No relevant conflicts of interest to declare.