A Rationale to Enhance the Response to Antimitotic Therapy in Acute Myeloid Leukemia

Abstract 1332

Acute myeloid leukemia (AML) is known to respond only moderately to antimitotic therapy while acute lymphoblastic leukemias can be efficiently targeted using spindle-disrupting agents. The underlying molecular cause for this clinical phenomenon is unknown. Recent evidence suggests that response to antimitotic therapy substantially depends on the stability of the critical mitotic regulator cyclin B. The ability to keep cyclin B expression levels stable during a mitotic block is associated with a good response leading to cell death in mitosis.

At the metaphase to anaphase transition of an unperturbed cell division, cyclin B is targeted for degradation by the anaphase-promoting complex/cyclosome (APC/C) to trigger chromosome separation. The spindle assembly checkpoint (SAC) is a surveillance mechanism to ensure that APC/C-mediated ubiquitylation is restricted to cells that show proper attachment of all chromosomes to a functional mitotic spindle. In case of spindle disruption or unattached chromosomes, the spindle checkpoint stays active which leads to interference with APC/C-dependent proteolysis of cyclin B blocking cells in prometaphase until every chromosome is attached to the mitotic spindle.

We recently developed a cell line-based reporter system which allows monitoring of cyclin B degradation under various conditions (Schnerch et al. Cell Cycle 2012). Here, we identified a pattern of slow degradation of cyclin B which continues through a mitotic block in case of chromosomal misalignment in unperturbed cell cycles. Remarkably, we also found prolonged slow degradation to trigger aberrant exit from mitosis in such cells giving rise to tetraploid cells. Therefore, a reduction in slow degradation appears as a promising rationale to foster a mitotic arrest and enhance cell death in mitosis during antimitotic therapy by preventing such mitotic slippage. We exposed our reporter cells to low concentrations of proteasome inhibitor during a spindle poison induced mitotic block to assess whether proteasome inhibition is capable of modulating slow degradation. Importantly, very low doses of proteasome inhibitor were sufficient to reduced the extent of cyclin B slow degradation during the mitotic block. Moreover, we demonstrate that low doses of proteasome inhibitor render the AML cell line Kasumi-1 responsive to low, non-disruptive concentrations of spindle poison (nocodazole and vincristine) leading to remarkable increases in the G2M-fraction. To the best of our knowledge there is no evidence so far that low doses of proteasome inhibitor exert antimitotic effects by interference with protein degradation during mitosis. Importantly, concentration of bortezomib of 1–2ng/ml (such as found in the serum of patients for up to 72h following administration of 1.3mg/m² bortezomib subcutaneously) were found to exert synergistic effects with antimitotic therapy. Increases in the percentage of G2M cells by 38% were observed in Kasumi-1 cells for the combination of vincristine and bortezomib. Based on these findings, we currently apply our system to probe combinations of proteasome inhibitor with modern tailored therapies that exert their antimitotic effects by activation of the SAC, such as inhibitors of the motor protein Eg5 or of the mitotic kinases Polo-like kinase 1 (Plk1) or Aurora A and B.

Using our cell line-based reporter system, we provide evidence in the in vitro setting that modulating slow degradation during antimitotic therapy by proteasome inhibition is a promising rationale to enhance the efficacy of antimitotic drugs. Drug concentrations used are based on published pharmacokinetics in humans and suggest feasibility of the drug combination in vivo. Our approach of targeted drug combinations may provide highly efficient treatment alternatives for patients that are not eligible for induction treatment.