Inhibition of Protein Kinase C beta by Enzastaurin (LY 317615) Induces Alterations of Key Regulators of Cell Cycle and Apoptosis in Mantle Cell Lymphoma and Synergizes with Chemotherapeutic Agents in a Sequence Dependent Manner.

Mantle Cell Lymphoma (MCL) is a subtype of malignant lymphoma, characterized by a poor clinical outcome, a median survival time of three years and almost no long-term survivors. In previous studies it could be shown that constitutive overexpression of NFκB pathway plays an important role in the pathogenesis of MCL. The protein kinase C (PKC) family is essential in cell signal regulation effecting cellular growth, proliferation and apoptosis. Previous studies in diffuse large B-cell lymphoma suggest that PKCβ overexpression enhances B-cell proliferation and is associated with poor clinical prognosis. The pivotal role of PKCβ in activation of NFκB renders it a promising therapeutic target in the therapy of MCL. Five MCL cell lines (GRANTA 519, HBL-2, Jeko-1, NCEB-1, Rec-1) and two haematological control cell lines (Jurkat, Karpas 422) were treated with Enzastaurin in a proliferation inhibiting dose of 10μM, defined in initial pilot experiments. Real-time PT-PCR and protein expression (Western blot) levels of various regulators of cell cycle and apoptosis were determined at various time points of enzastaurin treatment (0.5 to 12 hours). In addition analysis of cell cycle and apoptosis were performed by flow-cytometry. All, cell lines were also exposed to different doses of enzastaurin in combination with cytarabine, fludarabine or mitoxantrone and analysed for efficacy by evaluating the combination index. Inhibition of proliferation was achieved in all cell lines in a dose and time-dependent manner, however susceptibility varied strongly within the cell lines characterized by IC 50 (24 hours) ranging from 8,0 μM in Jeko-1 to > 50μM in GRANTA 519, Rec-1 and Karpas 422. Induction of apoptosis was detected in all cell lines according to susceptibility, and cell cycle analysis exhibits a moderate accumulation of and cells in the G2/M phase. Significant alterations of RNA expression was detected for BCL-2, cyclin D1 and p14 applying the Kruskal-Wallis-Test. Preliminary Western blot data confirm the altered levels of protein expressions. Synergistic effects of Enzastaurin combinations were sequence dependent, and resulted in a super-additive effect when the exposure of cells to cytostatic agents was followed by Enzastaurin treatment. No synergy effect was observed in combination with fludarabine. Our study revealed alteration of gene expression, critical for the regulation of cell cycle and apoptosis, after Enzastaurin treatment. Even more important, combination with specific cytostatic drugs demonstrated a synergistic efficacy in vitro. Thus, our data suggest that inhibition of PKCβ can overcome chemotherapy resistance by cellular survival mechanisms supporting the evaluation of such combinations in clinical trials.