RNA-Expression and Proteome Analysis Identify Complementary but Not Identical Molecular Targets in Enzastaurin-Treated Mantle Cell Lymphoma.

Protein kinase C beta (PKCbeta), a pivotal enzyme in B-cell signaling and survival, is over-expressed in most cases of mantle cell lymphoma (MCL) and results in activation of PI3K/AKT pathway. Enzastaurin, an oral serine/threonine kinase inhibitor, suppresses signaling through PKCbeta/PI3K/AKT pathways, induces apoptosis, reduces proliferation, and suppresses tumor-induced angiogenesis. Aim: To optimize the treatment options with this promising inhibitor the goal of this study was to elucidate the molecular pathways altered by Enzastaurin treatment in MCL.

Four documented MCL cell lines (Granta 519, HBL-2, Jeko-1, Rec-1) were harvested after 2-8h Enzastaurin exposure at a previously defined dose of 10μM and analyzed by RNA-array and proteome analysis as previously described (2D-polyacrylamide-gel-electrophoresis (2D-PAGE); Weinkauf 2009). Regulated molecules were mapped in a functional interaction network and candidates representing different pathways were verified by Western blotting.

Enzastaurin exposure led to significant reduction of cell viability in all cell lines (15-20%). This was also reflected in distinct alterations of the observed protein patterns in 2D-PAGE after enzastaurin exposure. Of a total of 977 concurrent protein spots 115 (12%) spots exhibited significantly (>3fold) altered protein levels after 4h of enzastaurin exposure. Mass spectrometry of 62 protein spots (39 increase; 23 decreased) identified 108 different candidate proteins, which were used to create a protein interaction network identifying the affected functional pathways. The results of the 2D-PAGE analysis were verified by Western blot in selected candidate proteins of apoptosis (VIM, PLEC1), DNA-repair (RAD50, PCNA, RFC1) and gene expression (EEF1D, SMC1A) pathways.

In parallel, RNA-expression array analysis identified 180 different genes regulated early after enzastaurin treatment. Again these genes were mapped to an interaction network, highlighting enzastaurin involvement in e.g. NFkB-, apoptosis and B-cell- and death receptor signaling pathways. Interestingly, the involved genes complemented the regulated proteins in the functional pathways. Network analysis of both screenings (2D-PAGE-based proteomics and RNA-expression array) classified the candidate molecules in functional groups, including DNA repair and replication (e.g. RAD50, PCNA), apoptosis (e.g.PSMC4, VIM), signal transduction (e.g. GRB2, EF1D) and gene expression/mRNA processing (e.g.EEF1D, SFRS7). Thus, combined analysis of both screening methods resulted in a more comprehensive network than each respective analysis.

Enzastaurin-treatment affects three main cellular control mechanisms as highlighted by two independent screening approaches (proteomics and expression array analysis). Interestingly, these two layers of molecular response (protein and RNA respectively) resulted in minimal overlap of identified molecules at this early (4h) time, but indicated common pathways nonetheless. Ongoing experiments now incorporate this knowledge to select optimal combination partners of enzastaurin.

Lilly Deutschland GmbH: Research Funding.