Supplementary Figure 3 from NUP98 Fusion Oncoproteins Promote Aneuploidy by Attenuating the Mitotic Spindle Checkpoint

PDF – 2287K, Supplementary Figure 3. Mitotic index in transfected and in nocodazole arrested HEK293 cells. A, Mitotic index of control untransfected, nocodazole-arrested HEK293 and U937 cells. Cells were labelled with a specific anti-phosphoSer10 antibody and analysed by flow citometry. B, Immunoblot analysis of the expression of the indicated proteins in HEK293 cells transiently transfected with expression constructs for NUP98, NUP98-HOXD13, NUP98-LOC, NUP98-HHEX, treated with nocodazole, and harvested at the indicated time points. An arrowhead indicates the NUP98-HOXD13 band just below that of endogenous NUP98. CyclinB1 (CCNB1), Geminin (GMN) and securin (SEC) amounts were detected C, HEK293 cells, transiently transfected with expression constructs for the indicated proteins, were labelled with a specific anti-phosphoSer10 antibody and analysed by flow citometry.

ARTICLE ABSTRACT

NUP98 is a recurrent fusion partner in chromosome translocations that cause acute myelogenous leukemia. NUP98, a nucleoporin, and its interaction partner Rae1, have been implicated in the control of chromosome segregation, but their mechanistic contributions to tumorigenesis have been unclear. Here, we show that expression of NUP98 fusion oncoproteins causes mitotic spindle defects and chromosome missegregation, correlating with the capability of NUP98 fusions to cause premature securin degradation and slippage from an unsatisfied spindle assembly checkpoint (SAC). NUP98 fusions, unlike wild-type NUP98, were found to physically interact with the anaphase promoting complex/cyclosome (APC/C)Cdc20 and to displace the BubR1 SAC component, suggesting a possible mechanistic basis for their interference with SAC function. In addition, NUP98 oncoproteins displayed a prolonged half-life in cells. We found that NUP98 stability is controlled by a PEST sequence, absent in NUP98 oncoproteins, whose deletion reproduced the aberrant SAC-interfering activity of NUP98 oncoproteins. Together, our findings suggest that NUP98 oncoproteins predispose myeloid cells to oncogenic transformation or malignant progression by promoting whole chromosome instability. Cancer Res; 74(4); 1079–90. ©2013 AACR.

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