Proteasome Inhibition Leads to Dephosphorylation and Downregulation of Protein Expression of Members of the Akt/mTOR Pathway In MCL

Mantle cell Lymphoma (MCL) is a distinct B-cell subtype characterized by the chromosomal translocation t(11;14)(q13;q32), an especially poor clinical outcome and low response to chemotherapy. The proteasome inhibitor bortezomib is approved for treatment of relapsed and refractory MCL and achieves a response rate of 30–40%. However, little is known which molecules represent the critical targets of proteasome inhibition and how different regulators of cell cycle and apoptosis are affected. Bortezomib has been shown to inactivate the NFkB pathway in MCL, but recent findings indicate Bortezomib is also active in a proteasome –independent manner suggesting Bortezomib targets multiple pathways.

Four MCL cell lines (HBL2, Granta 519, Jeko-1, NCEB-1), two CLL cell lines (Mec1, Mec2) and two hematological control cell lines (Jurkat, Karpas 422) were exposed to Bortezomib at a previously defined cytotoxic concentration (25nmol). Western blot and mRNA analysis were performed for various members of the PI3K/Akt/mTOR and the MEK/ERK pathway after 24h Bortezomib exposure. Results were compared to cell proliferation (WST1, trypan blue staining), induction of apoptosis (Annexin V PE/7-AAD staining) and cell cycle data (FACS).

Western blot analysis revealed reduced phosphorylation of Akt at Ser473 in all cell lines while autophosphorylation of mTOR at Ser2481 was completely downregulated only in the susceptible cell lines. In addition further members of the mTOR pathway were affected by bortezomib treatment. Dephosphorylation of the 4EBP1, downregulation of p70S6 protein expression was detected in all cell lines whereas EIF4E dephosphorylation was only observed in the sensitive MCL cell lines. In contrast Bortezomib did not affect members of the MEK/ERK pathway (MEK1/2, p42/44MAPK). Interestingly the mRNA and protein expression profile of CCND1 were differently altered suggesting an involvement of bortezomib in the regulation of translation initiation. This data were also confirmed by microarray analysis.

In this study Bortezomib treatment was shown to target the Akt/mTOR pathway especially by dephosphorylation of the translation initiation factor EIF4E and other molecules of this signal pathway. This knowledge will play a crucial role in the development of future combinations of biologicals to target the molecular pathogenesis of MCL.

Dreyling:Johnson & Johnson: support of investigators initial trials.