Enzastaurin Treatment Affects Multiple Regulatory Pathways at Transcriptome and Cellular Proteome Level of Mantle Cell Lymphoma

The protein kinase C beta inhibitor enzastaurin is one of the promising molecular targeted approaches currently investigated in mantle cell lymphoma (MCL), a disease still characterized by a dismal long term prognosis.

Four well characterized MCL cell lines (Granta 519, HBL-2, Jeko-1 and Rec-1) as well as three patient samples were exposed to enzastaurin at a previously defined dose (10 μM). Cell viability as well as cell cycle activity were analyzed by tryphan blue exclusion test and flow cytometry, respectively, after 24 and 48 hours. To dissect the regulatory processes targeted by enzastaurin, the panel of MCL cell lines was screened on both protein and RNA expression levels (2D-gel electrophoresis and mass spectrometric peptide fingerprint analysis and Affymetrix microarray) after 4h enzastaurin treatment.

Enzastaurin in vitro resulted in a reduced viability and cell proliferation by 15–20% after 24h in cell lines and 9–20% in primary patient samples after 48h. This effect was related to a G2/M block of cell cycle and induction of apoptosis. Based on the proteome and transcriptome analysis of early alterations, only HSPD1 was affected on both regulation levels. Nonetheless, combined analysis of alterations on both, protein and RNA expression levels, resulted in identification of common signal pathways characterizing a more comprehensive network of affected molecular interactions mapping to distinct canonical pathways and defined cellular functions. Indicated canonical pathways included ‘calcium signalling', (CAMKK2, HDAC5, HDAC9, TP63) ‘calcium induced T-lymphocyte apoptosis' (MEF2D, NR4A1, PRKCG, TRA@), ‘NFkB signalling' (KRAS, MAP3K8, TNFAIP3, TNFRS17) and ‘molecular mechanisms of cancer' (APAF1, CDKN2D, FOS, PAK6), whereas the top ranking cellular functions were ‘cellular growth and proliferation' (CCNG2, EIF4E, PDIA3, TOP1, TPM1,), ‘cell death' (BCL6, EEF1D, PAK6, RAD50), ‘cell cycle'(AKAP9, BMF, CUL5, GADD45B, PDIA3), ‘cellular development' (APAF1, GAS7, ID1, PAX8) and ‘gene expression' (ABCG1, HOXB4, LMO4, PIM1). Alterations of these pathways were confirmed by Western Blot analysis of selected candidate proteins marker proteins of the regulated pathways.

In summary, the combined approach of RNA and protein analysis revealed the targeted signal pathways after Enzastaurin exposure. These data will allow a more rationally designed combination of biologicals to finally improve the clinical outcome of MCL.

Dreyling:Eli Lilly: Support of in vitro studies of Enzostaurin in MCL.