Synergistic Antilymphoma Effect of Protein Kinase C Beta (PKCβ) and mTOR Inhibition in Mantle Cell Lymphoma.

The protein kinase C (PKC) family of enzymes are serine/threonine kinases essential to the cell signal cascades effecting cellular growth, proliferation and apoptosis. Accordingly PKCβ overexpression correlates with poor clinical prognosis in diffuse large cell lymphoma. The pivotal role of PKCb in neoplastic transformation renders it a potential therapeutic target in the therapy of hematologic malignancies.

To determine drugs which are efficiently inhibiting cell proliferation in combination with enzastaurin in MCL.

Five MCL cell lines (HBL-2, GRANTA 519, Jeko-1, Z138, Rec-1) and patient samples were cultured in the presence of LY317615 (PKCb inhibitor), rapamycin (mTOR inhibitor) and LY294002 (PI3K inhibitor). Cell proliferation and viability was assessed by cell count and WST-1 proliferation assay. Analysis of cell cycle profile and apoptosis was performed by flow cytometry (PI and Annexin V FITC staining). mRNA expression was measured before and after treatment (8h) by microarray and real time PCR in cell lines. Protein expression was analysed by Western blot.

In a panel of mantle cell lymphoma cell lines, with IC50 values ranging from 2 to 5 microM for enzastaurin treatment, a refractory to enzastaurin cell line (Rec-1) was characterized. Treatment of the cell lines with enzastaurin induced apoptosis and lead to accumulation of cells in the G2, M phase in susceptible cell lines (Hbl-2, Jeko-1), whereas cell cycle profile remained unaltered in the refractory cell line (Rec-1).

While enzastaurin induced increased phosphorylation of mTOR and MEK and decrease of p90^RSK phosphorylation in all MCL cell lines, mTOR phosphorylation was twice as high in the refractory cell line (Rec-1). In line with this observation the combination of enzastaurin with rapamycin lead to a synergistic effect on the inhibition of cell proliferation in the Rec-1 cell line as well as in an additional MCLpatient sample. Protein expression levels (low CCND1, phAkt, php90^RSK, phPDK) achieved in Rec-1 after treatment with enzastaurin were also characteristic for the cell lines more sensitive to rapamycin. In contrast in some cell lines combination of enzastaurin and the PI3K inhibitor (LY294002) displayed antagonism. Further mRNAand proteinexpression analysis of patient samples are ongoing to determine molecular predictors of drug sensitivity.

In our study a combination of rapamycin and enzastaurin acted synergistically in MCL cell lines and a patient samples whereas the combination with a PI3K inhibitor displayed partial antagonism. Based on this results we have identified the underlying signal pathways to develop new synergistic molecular combinations in MCL.

Hutter:Lilly Deutschland GmbH: Research Funding. Zimmermann:Lilly Deutschland GmbH: Research Funding. Rieken:Lilly Deutschland GmbH: Research Funding. Weinkauf:Lilly Deutschland GmbH: Research Funding. Dreyling:Lilly Deutschland GmbH: Research Funding.