Genome-Wide Crispr-Cas9 Screen Identifies Functionally Relevant Micro-RNAs in FLT3-ITD+ AML
Authors: Wallace Jared , Kagele Dominique , Hu Ruozhen , Runtsch Marah , Alexander Margaret , Huffaker Thomas , Mosbruger Timothy , Dahlem Timothy , Stephens W. Zac , Rao Dinesh S. , Miles Rodney R. , Round June , O'Connell Ryan M. 
Published: October 2019

Abstract
Mammalian microRNA expression is dysregulated in many human cancers, including hematologic malignancies. MicroRNAs are small non-coding RNAs that repress their target genes by binding to the 3' UTR sites in their respective mRNA targets, leading to transcript degradation and/or preventing translation. MicroRNAs play a variety of physiologic roles within the hematopoietic compartment, and a few microRNAs have even been shown to drive leukemogenesis when deleted or expressed at abnormally high levels. Among hematologic malignancies, acute myeloid leukemia (AML) carries a particularly poor prognosis, leading to over 10,000 deaths each year. The most common genetic aberration in AML is a gain-of-function mutation in the FMS-like tyrosine kinase 3 (FLT3) receptor. FLT3 internal tandem duplication (ITD) occurs in ~25% of all AML diagnoses, and confers a poor prognosis due to increased cellular proliferation and survival of the hematopoietic stem and progenitor cells (HSPCs). MicroRNA expression has been shown to be highly dysregulated in FLT3-ITD+ AML, however the functional relevance of many of these microRNAs on leukemic phenotypes remains unclear. In this study, we took an unbiased approach to determine which microRNAs, and which of their putative targets, are involved in FLT3-ITD+ AML cellular growth.
Using CRISPR-Cas9 technology, we performed a global loss-of-function screen to simultaneously test the functions of individual microRNAs and protein-coding genes during the growth of FLT3-ITD+ leukemia cells (MV4-11 cells) over a 23-day time course. By comparing library representation from genomic DNA isolated from the final time point versus the initial time point, we were able to identify a number of protein-coding gene and microRNA candidates that either promoted or suppressed FLT3-ITD+ AML cellular growth.
Our screen identified both evolutionarily conserved and non-conserved human microRNAs that function to suppress or promote FLT3-ITD+ AML cellular growth, revealing that microRNAs are extensively integrated into the molecular networks that control tumor cell physiology. We also performed anti-correlation functional profiling to predict relevant microRNA-tumor suppressor gene or microRNA-oncogene interactions in FLT3-ITD+ cells. We validated one of our targets, miR-155, as a critical regulator of FLT3-ITD+ AML cell growth in vitro. miR-155 knockout cells displayed a competitive growth disadvantage compared to miR-155 wild type cells. Further analysis revealed that deletion of miR-155 in MV4-11 cells led to decreased STAT5 activation, a key signaling intermediate known to promote cell survival and proliferation in FLT3-ITD+ AML. Finally, we found that miR-155 promotes FLT3-ITD-mediated myeloproliferation in vivo. FLT3-ITD miR-155-/- mice exhibited decreased myeloid expansion in the bone marrow, reduced splenomegaly, and decreased peripheral blood monocytosis compared to their FLT3-ITD miR-155+/+ counterparts. This phenotype was attributed to miR-155's role in promoting proliferation of the HSPC and myeloid progenitor cell compartments in the bone marrow.
Our CRISPR-Cas9 screen identified a subset of microRNAs that regulate FLT3-ITD+ cell growth, and extensively validated miR-155 as a promoter of FLT3-ITD+ cell proliferation. These findings were validated both in vitro and in vivo, and suggest that miR-155 inhibitors may warrant clinical consideration as therapeutics in FLT3-ITD+ AML. Taken together, our study describes a powerful genetic approach by which the function of individual microRNAs can be assessed on a global level, and its use could rapidly advance our understanding of how microRNAs contribute to human hematological diseases.

Disclosures
No relevant conflicts of interest to declare.