Enzastaurin Induces Apoptosis and Cell Cycle Arrest in B-Cell Acute Lymphoblastic Leukemia Cell Lines Through AKT Pathway Inhibition and ß-Catenin Accumulation

Abstract 1350

Precursor B-Cell acute lymphoblastic leukemia (B-ALL) is the most common leukemia in children and accounts for 20% of acute leukemia in adults. The intensive induction–consolidation–maintenance therapeutic regimens used currently have improved the 5-year disease free survival to around 80% in children and to 25%-40% in adults. The poorer response in adults is due to the inability to tolerate the intensive chemotherapy, and to the biology of adult disease which is associated with poor-risk prognostic factors. In the present era of target-specific therapy, protein kinase C beta (PKCß) targeting arose as a new, promising, and well-tolerated treatment strategy for a variety of neoplasms, especially in B-cell malignancies. The most frequently examined drug candidate to date is enzastaurin (LY317615.HCl) (ENZ), an acyclic bisindolylmaleimide that is orally administered and selectively inhibits PKCß. PKCß plays a major role in B-cell receptor signaling, but studies describing the role of PKCß in B-ALL are primitive. In the present study, we investigate the effect of ENZ on a variety of B-ALL cells representing the wide spectrum of the disease.

Seven B-ALL cell lines were studied: RS4;11 and SEM-K2 [both Pro-B ALL with t(4;11)(q21;q23)], TOM-1 and SUP-B15 [both Ph-positive Pro-B ALL with t(9;22)(q34;q11)], HB-1119 [Pre-B ALL with t(11;19)(q23;p13)], NALM-6 [Pre-B ALL with t(5;12)(q33;p13)], and Reh [Pre-B ALL with t(12;21)(p13;q22)]. Cells were tested against serial dilutions of ENZ (final concentrations: 0.5–20μM) for 24, 48, and 72 hours in flat bottom 96-well plates. MTS assay was performed to quantify cell viability. ENZ induced a dose and time-dependent cell growth inhibition in B-ALL cell lines. RS4;11, SEM-K2, and HB-1119 (all with translocations involving the MLL gene) showed the greatest sensitivity to ENZ, with statistically significant cell growth inhibition starting at 1 μM, a concentration easily achieved in-vivo. TOM-1 and SUP-B15, both Ph-positive ALL, showed the lowest sensitivity to ENZ. The mechanism of ENZ cell growth inhibition was shown by flow cytometric TUNEL assay to involve apoptotic induction and cell cycle inhibition. Because of its relatively high sensitivity to inhibition among B-ALL cells, RS4;11 was selected for further analysis of the effect of ENZ on phosphorylation of AKT and its downstream target GSK3ß. RS4;11 cells were treated with the corresponding IC50 of ENZ for 0.5, 1, 2, 4, 24, and 48 hours. Treatment resulted in a time-dependent loss of AKT phosphorylation, at both ser473 and thr308, and a decrease in GSK3ß phosphorylation starting after 30 minutes and continuing to 48 hours. No effect on total AKT and GSK3ß was observed. By activating GSK3ß, its downstream target ß-catenin was expected to be diminished secondary to phosphorylation and proteasomal degradation. Surprisingly, ENZ induced a rapid and sustained ß-catenin accumulation, in both its nuclear and cytoplasmic forms. This was explained by a transient loss of ß-catenin phosphorylation at ser33-37; no effect on the proteasome activity was observed. Similar effect on total and phosphorylated ß-catenin was observed in all other cell lines. ß-catenin represents a central component of Wnt/ß-catenin canonical pathway which is found to be implicated in ALL pathogenesis. To investigate the effect of ENZ on Wnt/ß-catenin pathway, total RNA (1 μg) from RS4;11 treated for 24 hours with ENZ was profiled on RT² Profiler™ PCR Array Human WNT Signaling Pathway (SABiosciences) and compared to untreated control. There were 8 genes whose expression changed >3-fold, most prominently c-Myc, c-Jun, and several genes encoding Wnt proteins. This was confirmed by western blot analysis showing that treatment with ENZ resulted in decreased c-Myc and increased c-Jun proteins expression. The latter showed a preliminary effect on p73, a p53 homologue, and is a subject for further investigation.

These results indicate that PKCß plays an important role in the malignant process in B-cell ALL, and suggest that ENZ should be considered as a potential treatment, whether in combination or as a single agent monotherapy. Ongoing studies in our lab will detail the mechanism of PKCß inhibition, explain the contribution of ß-catenin accumulation to the cytotoxic effect of ENZ, and possible relationships between PKCß signaling and 11q23 translocation.