Targeting CDK4 in Mantle Cell Lymphoma (MCL) Cell Lines by Specific Lentiviral shRNA Mediated Knockdown Has Profound Effects on Cell Growth and Cell Cycle but Minimal Effects on Apoptosis.

Introduction: The hallmark of MCL is overexpression of cyclin D1 due to the t(11;14). Cyclin D1 accelerates the transition of lymphoma cells from G1 to S phase by binding the cyclin-dependent kinase 4 (CDK4), and inactivating the retinoblastoma protein. Due to the importance of cyclin D1-CDK4 complexes in the control of the cell cycle, CDK4 has become a novel and attractive therapeutic target in MCL. Flavopiridol is a pharmacologically derived CDK inhibitor, which is in phase II trial in relapsed MCL patients. The aim of this study was to investigate the effects of silencing CDK4 expression in MCL using highly specific CDK4-shRNA, and compare the results with known effects of flavopiridol and other CDK inhibitors.

Material and methods: A highly efficient CDK4-shRNA was identified using a LacZ-cyclin D1 fusion gene reporter system in HEK293T cells. This shRNA was cloned into a lentiviral transfer vector carrying GFP as a reporter gene, which enables the detection of infected cells by FACS analysis. Four MCL cell lines were analyzed (Granta 519, Jeko-1, Rec-1, Z-138), using appropriate controls. Western Blot analysis and qRT-PCR were performed to quantify the knockdown effect. The effect of CDK4 knockdown on proliferation, cell cycle, and viability was analyzed by MTT assay and FACS analysis.

Results: In MCL cell lines with high CDK4 levels (Jeko-1, Rec-1, Z-138), profound effects on cell growth and cell cycle were observed with only minor effects in apoptosis. The cells showed 40% growth retardation compared to control cells after seven days of infection. Additionally, a clear shift from the S phase to the G1 phase in the cell cycle was observed as compared to control cells (S phase decreased by 23% and G1 increased by 28%). In contrast, only minimal apoptosis was induced (11% increase compared to control cells). CDK4-shRNA infected cells showed a strong induction of cyclin D2 mRNA (5,6 fold on average). Additionally, Granta 519 and Z-138 cell lines showed an increase in CDK6 mRNA. The expression of cyclin D1 mRNA and protein and cyclin E protein remained unchanged or was slightly induced. No changes were observed in the protein expression of cyclin D3, p27Kip1 and CDK2.

Conclusions: Our data showed that the specific CDK4 downregulation induces important growth retardation but not significant apoptosis. The reported effects after Flavopiridol treatment including downregulation of cyclin D1 and D3, as well as the induction of apoptosis, were not observed after specific CDK4 knockdown, and indicate CDK4-independent effects of this CDK inhibitor. Taken together, targeting specifically CDK4 alone is likely to have less effect on MCL than broader kinase inhibitors like Flavopiridol, used either as a single drug or in combination with conventional chemotherapy.