The mechanism by which cells decide to skip mitosis to become polyploid is 
largely undefined. Here we used a high-content image-based screen to identify 
small-molecule probes that induce polyploidization of megakaryocytic leukemia 
cells and serve as perturbagens to help understand this process. Our study 
implicates five networks of kinases that regulate the switch to polyploidy. 
Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased 
polyploidization, mature cell-surface marker expression, and apoptosis of 
malignant megakaryocytes. An integrated target identification approach employing 
proteomic and shRNA screening revealed that a major target of diMF is Aurora 
kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective 
inhibitor of AURKA, induced polyploidization and expression of mature 
megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and 
displayed potent anti-AMKL activity in vivo. Our findings provide a rationale to 
support clinical trials of MLN8237 and other inducers of polyploidization and 
differentiation in AMKL.