Genome-Wide RNAi Screens for Novel Regulators of Acute Myeloid Leukemia

Abstract

Acute myeloid leukemia (AML) is a heterogeneous disease with complex molecular mechanisms. Recent advent of genomic technologies, such as copy number profiling, whole genome sequencing, and gene expression profiling has accumulated a plethora of large-scale data in AML cell lines and patient samples. However, the functional relevance of most genes identified by these methods has yet to be determined. To systematically characterize the genetic requirement in AML, we conducted genome-wide shRNA screens in 17 AML cell lines in parallel with 199 cell lines of other cancer types. We identified over 150 genes that were required for proliferation specifically by AML, but not other cancer cell lines. We further interrogated the requirements of primary screen hits in vivo with a secondary screen in a xenotransplantation model driven by the MLL-AF9 oncogenic fusion. Integrating both of the RNAi screens and additional gene expression data, we identified transcription factor ZEB2 as a top candidate for regulating AML proliferation. In human AML cells, ZEB2 inhibition impairs proliferation and promotes granulocytic differentiation. Mechanistically, we showed that ZEB2 interacts with the CtBP co-repressor complex, and transcriptionally represses genes involved in cell adhesion and migration. ZEB2’s relevance in AML is further demonstrated by its overexpression in MLL-rearranged AML, and by the epigenetic silencing of its negative regulators, miR-200 family microRNAs, in AML. Our results extend the role of ZEB2 beyond regulating epithelial-mesenchymal transition, and establish ZEB2 as a novel regulator of AML proliferation and differentiation.

MicroRNA-like off-target effect is a major caveat of RNAi screens, which often leads to false positive discoveries. However, systematic analysis of off-target effects in large-scale RNAi screen data can also lead to the discovery of microRNAs with functional relevance. By analyzing the off-target effects in our AML screen, we identified several microRNAs as candidate suppressors for AML proliferation. We show that miR-105, miR-140, miR-501, and miR-532 are novel regulators of the myeloid oncogene MYB. In particular, miR-105 inhibits AML cell growth and miR-532 is associated with myeloid differentiation. The combination of the ZEB2 and microRNA work emphasizes the power of RNAi screens in the exploration of novel cancer regulators.