Proteomic Analysis of Pkc-Beta Inhibitor Enzastaurin-Treated Mantle Cell Lymphoma

Background: Activation of the PI3K/AKT pathway is involved in pathogenesis of MCL and accordingly, protein kinase C beta (PKCbeta), a pivotal enzyme in B-cell signalling and survival, is over- expressed in most cases of mantle cell lymphoma (MCL). Enzastaurin, an oral serine/threonine kinase inhibitor, suppresses signalling through PKCbeta/PI3K/AKT pathways, induces apoptosis, reduces proliferation, and suppresses tumor-induced angiogenesis. Recently, a phase II study showed that enzastaurin is able to prolong the progression-free survival in a subset of relapsed MCL (Morschhauser et al, Ann Oncol 2008).

Methods: Four MCL cell lines (GRANTA 519, HBL-2, Jeko-1, Rec-1) were treated with Enzastaurin at a proliferation inhibiting dose of 10μM, defined in previous pilot experiments. After 2–8h of treatment, cells were harvested and a two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE)-based protein screening was performed.

Results: Already 1h after treatment, distinct alterations of the protein pattern were recognized in the 2D-PAGE gels of all cell lines. 115 (12%) of 977 concurrent protein spots exhibited significantly (>3fold) altered protein levels after 4h of enzastaurin exposure, i.e. increased (57 spots; 5,8%) or decreased (58 spots; 5,9%) protein expression, and after 8h the percentage of affected protein spots further increased up to 22% of all concurrent protein spots, due to decreased protein levels. Based on previously defined selection criteria (Weinkauf et al, Electrophoresis 2007) 62 differentially expressed protein spots at 4h (increased/decreased expression: 39/23 spots) were chosen for mass spectrometric identification. Mass spectrometry identified 108 different candidate proteins with significant identification confidence (p<0.05). 29 candidate proteins were detected in multiple protein spots, while 79 candidate proteins were identified in only single protein spots. The identified candidate proteins could be classified in distinct cell programs, including DNA repair and cell division, cytoskeleton (centrosome and spindle), apoptosis, signal transduction and transcription factors. Interestingly, some of the identified candidates had been also previously identified after proteasome inhibition by bortezomib, e.g. ENOA, TOP1, CE290, SMC1A, HSP60 (CH60), ATPB, KAD5 and TPIS.

Conclusion: The identified candidate proteins are currently confirmed in MCL patient samples. Characterization of essential signal pathways after Enzastaurin exposure will not only expand the understanding of the molecular pathogenesis of MCL, but also hint towards promising targets for new molecular therapies.