RAD001 (everolimus) down-regulates cyclin D3 and c-Myc and is particularly effective in the treatment of aggressive B-cell lymphomas

17573 Background: Aggressive B-cell lymphoma is recently found to be associated with constitutive activation of the PI3K/Akt pathway, and thereby is relatively resistant to apoptosis. Furthermore, activation of the PI3K/Akt signaling pathway can result in the upregulation of cyclin D3 and c-Myc through inhibition of the cap-independent RNA translation. Since mTOR is closely associated with the regulation of the translation process, and is a downstream mediator of the PI3K/Akt signalling pathway, this study aimed to examine if RAD001, an inhibitor of mTOR, can be effective in the treatment of aggressive B-cell lymphoma. Methods: Pfeiffer (diffuse large cell lymphoma), Ramos (EBV (−) Burkitt’s lymphoma), Raji (EBV (+) Burkitt’s lymphoma), and MC116 (EBV (−) undifferentiated lymphoma) cell lines were used in this study. Expression of pAkt, p70S6 kinase, pp70 S6 kinase, 4E-BP1, p4E-BP1, cyclin D3, and c-Myc was measured by immunoblotting. Results: Akt was constitutively activated in all four lymphoma cell lines. Downstream effectors of PI3/Akt signaling pathway, including p70S6 kinase and 4E-BP1, were also phosphorylated in these lymphoma cell lines. RAD001 downregulated p70S6 kinase and 4E-BP1, and the IC50s of growth suppression ranged from 5 to 50 nM, without significant difference between EBV (+) and EBV (−) cell lines. The IC50s of these lymphoma cell lines appeared to be much lower than those obtained from the carcinoma cell lines, suggesting that lymphomas may be particularly sensitive to growth inhibition by RAD001. RAD001 blocked cell cycle progression in G0/G1 phase in all four lymphoma cell lines while there was no significant increase in sub-G1 phases, suggesting that apoptosis was not the major mechanism of RAD001-induced growth inhibition. In addition, in parallel with the RAD001-induced growth inhibition, a dose-dependent down-regulation of the expression of cyclin D3 and c-Myc was demonstrated. Conclusions: The mechanism of action is at least partly due to downregulation of cyclin D3 and c-Myc, and subsequent G0/G1 block. Since overexpression of cyclin D3 and c-Myc is also closely associated with chemoresistance of aggressive B-cell lymphoma, RAD001 may be a useful adjunct in the treatment of this group of tumors. No significant financial relationships to disclose.