The Selective Protein Kinase CB Inhibitor, Enzastaurin, Induces In Vitro and In Vivo Antitumor Activity in Waldenstrom’s Macroglobulinemia.

Background: Waldenstrom’s Macroglobulinemia is an incurable lymphoplasmacytic lymphoma with limited options of therapy. We have previously demonstrated upregulation of PKCβ protein in WM using protein array techniques, and confirmed increased expression in WM using immunohistochemistry. PKCβ regulates cell survival and growth, as well as migration and homing in many B-cell malignancies. We therefore hypothesized that inhibition of PKCβ will induce cytotoxicity in WM.

Methods: In this study, we examined the effect of serial dilutions of the PKCβ enzastaurin (2.5 uM to 20 uM) on WM cell lines (BCWM1 and WM-WSU), IgM secreting low-grade lymphoma cell lines (MEC-1, RL), as well as primary CD19+ WM cells and WM cells adherent to bone marrow stromal cells (BMSCs), which induce resistance to conventional therapy. Cytotoxicity was measured by MTT assay and inhibition of cell proliferation was determined by thymidine uptake assay. Apoptosis was measured by flow cytometry using Annexin V and DAPI staining at 48 h. Cell DNA content analysis was performed using DAPI staining on fresh cells. Cell signaling pathways targeted by enzastaurin were determined using immunoblotting at 6 h (2.5 to 10 uM) and at 7.5 uM (10 min to 12 h). The effect of enzastaurin in vivo was determined using a subcutaneous WM model in SCID mice. Enzastaurin was given by oral gavage (80mg/kg twice daily).

Results: Enzastaurin demonstrated time and dose-dependent inhibition of PKCβ in WM cells. It induced a significant decrease of proliferation at 24 and 48 h in all cell lines tested with an IC50 of 2.5 to 10 uM, even in the presence of DOPPA, a specific PKCβ stimulator. Similar effects were demonstrated in primary CD19+ WM cells, with no cytotoxicity on peripheral blood mononuclear cells indicating selective toxicity on malignant cells. Enzastaurin induced dose-dependent apoptosis at 24 and 48 h with induction of caspases 3, 8, 9 and PARP cleavage as well as a decrease in Bcl-xL. Analysis of cell DNA content confirmed apoptosis at low doses of enzastaurin (5 uM). To further determine the mechanism of action of enzastaurin in WM, we examined downstream molecules. It significantly inhibited AKT phosphorylation and AKT kinase activity, as determined by inhibition of phosphorylation of GSKα/β fusion protein. In addition, enzastaurin inhibited p-MARCK, and ribosomal p-S6. Enzastaurin overcame resistance induced by co-culture of WM cells with bone marrow stromal cells. In addition, enzastaurin (2.5 to 5 uM) in combination with bortezomib (2.5 to 10 nM), another active agent in WM, demonstrated strong synergistic activity using the Calcusyn software for synergy. Given that PKCβ regulates migration and homing of B-cells, we next determined the effect of enzastaurin on in vitro migration of WM cells. In the transwell migration assay, enzastaurin inhibited migration in a dose-dependent fashion (p=0.041). Finally, in vivo animal studies demonstrated significant inhibition of WM tumor growth in the enzastaurin treated mice (n=11), compared to control mice (n=8) (p=0.028).

Conclusion: Enzastaurin has significant antitumor activity in WM in vitro and in vivo, providing the framework for clinical trials evaluating enzastaurin as a new therapeutic agent in patients with WM.