A Kinome shRNA Screen to Identify Pathways That Regulate Megakaryocyte Polyploidization and New Targets for Differentiation Therapy

Abstract 89

Megakaryocytes undergo a unique mode of cell cycle called endomitosis, which is characterized by repeated rounds of DNA synthesis without cell division. Normally, polyploidization is tightly coupled with expression of megakaryocyte specific genes and terminal differentiation. Although there have been several reports of differences between the proliferative and endomitotic cell cycles, the precise mechanisms that regulate the switch to polyploidization and terminal differentiation remain unknown. To identify genes that play a role in this process, we performed a high throughput shRNA screen assaying for genes whose decreased expression induce polyploidization in CMK megakaryocytic leukemia cell line. We used a kinase biased library of shRNAs, reasoning that targeting the kinome would identify pathways or complexes important for this switch. After transduction of with the lentiviral shRNA library, CMK cells were selected with puromycin, fixed and stained with Hoechst dye. A high content imaging assay using CellProfiler software was employed to analyze the DNA content of microscopic images of cells. By this approach, we screened 775 genes, 650 encoding kinases, with at least 5 hairpins per gene. In parallel, we performed an otherwise identical screen (i.e., modifier screen) that included sub-maximal doses of dimethylfasudil, a drug that we have previously shown to promote megakaryocyte polyploidization. To reduce the off-target effects of shRNA, we only considered genes that scored with two or more shRNAs. In comparison to non-targeted shRNA controls, we considered the top 5% of genes in either the direct or modifier screens to be hits. By this analysis, we identified 71 genes whose loss promoted polyploidization. Pathway analysis revealed that the majority of these genes clustered into five groups. Among these clusters, one was notable for containing AURKA (aurora kinase A), AURKB (aurora kinase B), CCNB1 (cyclin B1), CDK2 and PLK1 (polo-like kinase). The screen revealed that knockdown of Aurora B kinase significantly increased polyploidization in the absence of diMF (p<0.001), but that knockdown of Aurora A kinase only increased polyploidization when diMF was included (p<0.01). Of note, although several studies have examined the expression and function of AURKB in polyploidization, its role in human versus mouse megakaryopoiesis is controversial. Moreover, whether inhibition of Aurora kinases would be a useful therapy for megakaryocytic leukemias, characterized by defects in polyploidization and terminal differentiation, has not been addressed. We discovered that MLN8237, a selective Aurora kinase A inhibitor, and AZD1152, a selective Aurora kinase B inhibitor dose dependently inhibited proliferation while inducing polyploidization, upregulation of megakaryocyte specific lineage markers, and apoptosis of CMK cells and the 6133-MPL murine cell line, which expreses the AMKL fusion protein OTT-MAL and the W515L constitutively active allele of MPL. MLN8237 and AZD1152 induced polyploidization with an EC50 of 22 nM and 210 nM in CMK cells respectively. Both compounds also induced robust polyploidization of human and mouse primary megakaryocytes cultured ex vivo. At the doses utilized, neither drug induced polyploidization of the non-megakaryocyte fractions of the ex vivo cultures, indicating that these inhibitors are selective for megakaryocytes at the doses used in this study. Finally, since we found that MLN8237 was a more potent inducer of polyploidization, we next assayed the ability of the compound to block progression of AMKL in an animal model. MLN8237 significantly increased the survival of 6133-MPL transplanted mice compared to vehicle (p=0.02), demonstrating the anti-leukemic activity of this small molecule. Together, our data suggest that Aurora kinase inhibitors may be useful as differentiation therapeutic agents for AMKL.