Canonical NF-kB Pathway Is Not Constitutively Activated and May Not Be the Major Bortezomib Target in Mantle Cell Lymphoma (MCL).

Bortezomib, a proteasome inhibitor, has promising activity in MCL. Bortezomib mechanism of action is complex since multiple proteins and cellular pathways are regulated by proteasome degradation. By blocking the degradation of IKBa, the canonical NF-kB pathway is one of the major Bortezomib target in lymphoid malignancies such as multiple myeloma. NF-kB activation has not been extensively assessed in other lymphoid malignancies such as MCL. NF-kB complexes (heterodimers P50/P65) were assessed by EMSA assays in REC GRANTA 519, NCEB, JEKO, JUN and UPN1 MCL cell-lines. EBV was present in 3/6 cell lines. P50/P65 complexes were detected in all EBV positive cell lines and only in 1/3 EBV negative cell-lines (JEKO). No nuclear P65 protein was detected by immunofluorescence or Western-blot analysis in the 2 other negative EBV cell lines (REC and UPN1). NF-kB transcriptional activity was measured by a luciferase-based reporter gene assay. Spontaneous NF-kB activity was low in UPN1, REC and JEKO compared to GRANTA 519 EBV positive cell-line (3 to 4 time lower) but can be strongly activated (up to 10 fold) using MEKK cotransfection assay. This suggest that NF-kB is functional, but not constitutively activated. Stable infections with Migr1-IRES-GFP IkBM (dominant negative IKB) or empty vector, were performed in GRANTA 519 EBV positive as well as in UPN1 cell line. No differences in proliferation or apoptosis were observed in UPN1 stably infected with IkBM or with the empty vector but GRANTA 519 EBV positive showed increased apoptosis and proliferation inhibition when infected with IkBM. Likewise, no P50/P65 heterodimers complexes were detected by EMSA in 4 patients with MCL. In vitro assays showed that UPN1 and JEKO cell lines had comparable sensitivities to Bortezomib than Multiple myeloma cell lines reported in the literature (IC 50: 6 nM and 12 nM respectively). This was also true for the 3 patients lymphoma cells assessed in vitro (12 nM). Taken together these results suggest that canonical NF-kB activation pathway is not constitutively active in EBV negative MCL cell lines and patients samples and could be associated with EBV infection in some MCL cell-lines. Therefore, this strongly suggests that Bortezomib target other molecules in MCL.