Spindle Checkpoint Insufficiency in Acute Myeloid Leukemia.
Authors: Schnerch Dominik , Felthaus Julia , Engelhardt Monika , Waesch Ralph M. 
Published: October 2019

Abstract
Chromosomal instability and aneuploidy are hallmarks of most human malignancies. Various mechanisms have been shown to give rise to numerical chromosome aberrations. Compromised function of the spindle assembly checkpoint (SAC) is generally regarded as one of the most powerful ways to drive genome instability. The SAC is a mitotic checkpoint mechanism ensuring the equal segregation of the mitotic chromosomes onto the developing daughter cells. Unfaithful mitotic surveillance by the SAC favors chromosomal misdistribution as error-prone chromosome attachment to the mitotic spindle does not induce a strong mitotic arrest by interference with anaphase promoting complex (APC)-dependent proteolysis. The APC is an important ubiquitin ligase that triggers the transition from mitosis into G1-phase by targeted proteolysis of mitotic regulators such as cyclin B and securin. The SAC prevents the proteolysis of those regulator proteins in the presence of mitotic aberrancies by inhibition of the APC. This leads to a delayed progression through mitosis and provides time to recover from defective chromosomal spindle attachment. SAC malfunction weakens the tight control on chromosome attachment and tension across the kinetochore favoring chromosomal misdistribution. We performed expression analyses of key proteins in SAC signaling in acute myeloid leukemia (AML). We found the SAC-components Bub1 and BubR1 to be down-regulated in most of the investigated AML cell lines. Functional assays revealed a defective mitotic arrest mechanism in comparison to SAC-competent cell lines after exposure to the microtubule disrupting agent nocodazole. This finding was accompanied by the observation of a decline in cyclin B and securin levels despite severe damage to the mitotic spindle induced by nocodazole. Expression of cyclin B and securin in the presence of spindle damage could be stabilized by proteasome inhibition. We established a shRNA-based model to evaluate the effects of BubR1- and/or Bub1-repression to levels found among AML cell lines to directly compare the Bub1/BubR1 knockdown phenotype with the investigated AML cell lines. Interestingly, BubR1 knockdown was sufficient to generate a phenotype resembling the behavior of our AML cell lines. Further experiments revealed a strong relation between premature degradation of cyclin B and the degree of BubR1 downregulation. Given the potent role of BubR1 in the generation of a mitotic arrest deficient phenotype, we addressed the BubR1 expression levels in a number of patients exhibiting karyotype abnormalities. Primary myeloid blast cells were stimulated with cytokines to force the largely resting cells into an actively dividing state. The maximum expression level of BubR1 in G2/M was used to define SAC-compentent and SAC-deficient populations. Strikingly, six out of eight (6/8) primary AML samples exhibited BubR1 expression patterns resembling the BubR1-knockdown model suggesting deficient mitotic surveillance in most of the primary AML samples. Since SAC deficiency is an important mechanism in creating numerical chromosomal aberrations and genetic instability, our findings underline a role for impaired SAC function in rise and progression of AML.