The Spindle Checkpoint Protein BubR1 Is Differentially Expressed in the Lymphatic and Myeloid Lineage and Is a Determinator of Response to Spindle Poisons

Spindle poisons disrupt the mitotic spindle thus leading to activation of the spindle assembly checkpoint (SAC). The SAC is a mitotic surveillance mechanism that can interfere with anaphase-promoting complex/cyclosome- (APC/C-) dependent proteolysis of key cell cycle regulators, such as securin and cyclin B, to delay cells at the metaphase to anaphase transition. The SAC protein BubR1 is a tumor suppressor with critical functions during mitosis and has also been shown to be important for the stablilization of cyclin B during interphase. We found this critical player to be downregulated in AML cell lines, primary AML blast populations and myeloblast-like murine 32D cells when compared to ALL cell lines and lymphoblast-like murine BaF3 cells. While myeloblast-like 32D cells are untransformed cells but also exhibit BubR1 repression we speculate that there might be a physiological role for a reduced BubR1 expression level in healthy myelopoiesis. However, in highly proliferative AML cells BubR1 repression might be a source of genetic instability due to a less efficient SAC-mediated interference with APC/C-dependent proteolysis in the presence of inaccuracies during mitosis.

Since repression of BubR1 is known to shorten the metaphase duration, we performed live-cell imaging of leukemia cells and found myeloblastic Kasumi-1 cells, expressing histone H2-GFP, to proceed faster through mitosis as compared to lymphoblastic DG-75 cells. While DG-75 cells exhibited a stable metaphase arrest upon spindle disruption using the spindle poison nocodazole, Kasumi-1 cells showed only a transient arrest, degraded securin and cyclin B and underwent sister-chromatid separation in the absence of a functional mitotic spindle. These findings suggest that the mitotic checkpoint is unable to properly interfere with APC/C-dependent proteolysis to prevent mitotic progression in myeloblastic leukemia upon treatment with spindle poisons. By using inducible retroviral reexpression of BubR1 and its downstream effector cyclin B we could enhance the ability of Kasumi-1 cells to accumulate in mitosis upon spindle disruption. Moreover, restoration of BubR1 led to higher cyclin B levels. Live-cell imaging analyses of Kasumi-1 cells, which expressed doxycyclin-inducible BubR1, revealed a prolonged metaphase, suggesting a more stringent control by the mitotic checkpoint when BubR1 expression is restored. Prolonged metaphase delays were also detected after reexpression of BubR1 when we challenged the cells with lower doses of spindle poisons suggesting that BubR1 is an important sensitizer for antimitotic therapies. Therefore, our finding of low BubR1 expression in AML provides an explanation for the poor response of myeloid leukemia to spindle poisons as compared to lymphoblastic leukemia. BubR1 has also been reported to be inactivated through promoter hypermethylation in various malignancies. The existence of a CpG island in the upstream region of the Bub1b locus (BubR1 coding sequence) tempted us to treat Kasumi-1 cells with the demethylating agent decitabine. Promotor demethylation led to an upregulation of BubR1 in mitotic AML cells providing evidence that BubR1 is a druggable target to enhance mitotic surveillance in AML cells.

Because mitotic therapies are widely used in the treatment of different malignancies, a further understanding of these processes might lead to a better understanding of cancer biology and improved therapeutic approaches.

No relevant conflicts of interest to declare.