Polo-Like Kinase-1 (Plk-1) Inhibitor BI 2536 Induces Mitotic Arrest and Apoptosis in Vivo: First Demonstration of Target Inhibition in the Bone Marrow of AML Patients

Background: BI 2536 is a potent and selective inhibitor of Plk-1, which plays a crucial role in the regulation of mitosis. Inhibition of Plk-1 leads to mitotic arrest and apoptosis. Therefore, Plk-1 inhibitors are currently in clinical testing as anti-proliferative agents in cancer patients. BI 2536, the first specific Plk-1 inhibitor in clinical testing, demonstrated strong anti-proliferative effects on AML cell lines in vitro and in in vivo models. To investigate target inhibition in the malignant cell, bone marrow samples from patients who participated in a Phase I/II study of BI 2536 single-agent therapy in elderly patients with relapsed or refractory AML were analyzed.

Methods: Pharmacodynamic analyses including immunocytochemistry (ICC) and flow cytometry (FACS) of bone marrow (BM) were performed to examine cell morphology, phosphorylation of histone H3 (phospho-H3), and induction of apoptosis. Samples were acquired before and 24 h after the first administration of BI 2536. FACS analysis included propidium iodide (PI)-FACS to determine the percentage of cells residing in various cell cycle stages (G0/1-, S- and G2/M-phases) and Annexin V-Cy5-staining for apoptosis. Immunocytochemistry included staining for phosphorylated histone H3 and TUNEL assay for apoptosis.

Results: At the time of analysis, data from 28 patients treated at doses in the range of 50 to 400 mg BI 2536 were available. BM taken after BI 2536 administration showed an increase of phospho-H3 positive cells as compared to baseline prior to treatment. There was a trend toward a positive correlation between phospho-H3 increase and increase in dosage of BI 2536 (p=0.16) when treatment at low doses (50 to 60 mg) of BI 2536 were compared to treatment at higher doses (100 to 400 mg). Furthermore, trends were observed that an increase of phospho-H3 is positively correlated with both a higher percentage of cells in G2/M and an increase in apoptotic cells. The typical morphology of cells in mitotic arrest could be demonstrated by ICC. Interestingly, when patients with progressive disease after one cycle (PD) were compared to patients with disease stabilization or response (nonPD), a statistically significant positive correlation between PD and increase in phospho-H3 compared to baseline was found (p=0.03). Also, there was a clear trend for an increase in the percentage of cells in G2/M phase and apoptosis in patients with PD compared to patients with nonPD. Preliminary evaluation of other disease characteristics including karyotype (normal vs complex), secondary AML, complete response in previous treatments, baseline value for blasts in the bone marrow or in the peripheral blood, did not reveal any signs of correlation with the clinical response to BI 2536 treatment.

Conclusion: BI 2536 treatment increases the number of cells in G2/M phase (as detected by FACS analysis and phospho-H3 staining) and the number of apoptotic cells within 24 h after administration. In line with the clinical observation of rapid blast reduction both in the BM and the peripheral blood, these findings indicate that BI 2536 induces mitotic arrest and apoptosis of the malignant target cells in AML patients. The biological meaning of the correlation between the BM findings and the clinical response to BI 2536 is unclear, but if substantiated by more data, these results may suggest a predictive value of BM examinations for the response to BI 2536.