Genome sequencing studies have shown that human malignancies often bear mutations in four or more driver genes1, but it is difficult to recapitulate this degree of genetic complexity in mouse models using conventional breeding. Here we use the CRISPR-Cas9 system of genome editing2–4 to overcome this limitation. By delivering combinations of small guide RNAs (sgRNAs) and Cas9 with a lentiviral vector, we modified up to five genes in a single mouse hematopoietic stem cell (HSC), leading to clonal outgrowth and myeloid malignancy. We thereby generated models of acute myeloid leukemia (AML) with cooperating mutations in genes encoding epigenetic modifiers, transcription factors, and mediators of cytokine signaling, recapitulating the combinations of mutations observed in the human disease. Our results suggest that lentivirus-delivered sgRNA:Cas9 genome editing should be useful to engineer a broad array of in vivo cancer models that better reflect the complexity of human disease.

The developmental switch from human fetal (g) to adult (b) hemoglobin represents a clinically important example of developmental gene regulation. The transcription factor BCL11A is a central mediator of g-globin silencing and hemoglobin switching. Here we determine chromatin occupancy of BCL11A at the human b-globin locus and other genomic regions in vivo by high-resolution chromatin immunoprecipitation (ChIP)-chip analysis. BCL11A binds the upstream locus control region (LCR), e-globin, and the intergenic regions between g-globin and d-globin genes. A chromosome conformation capture (3C) assay shows that BCL11A reconfigures the b-globin cluster by modulating chromosomal loop formation. We also show that BCL11A and the HMG-box-containing transcription factor SOX6 interact physically and functionally during erythroid maturation. BCL11A and SOX6 co-occupy the human b-globin cluster along with GATA1, and cooperate in silencing g-globin transcription in adult human erythroid progenitors. These findings collectively demonstrate that transcriptional silencing of g-globin genes by BCL11A involves long-range interactions and cooperation with SOX6. Our findings provide insight into the mechanism of BCL11A action and new clues for the developmental gene regulatory programs that function at the b-globin locus.[Keywords: Hemoglobin switching; fetal hemoglobin; BCL11A; transcription regulation] Supplemental material is available at http://www.genesdev.org.

SUMMARY Leukemia stem cells (LSCs) have the capacity to self-renew and propagate disease upon serial transplantation in animal models, and elimination of this cell population is required for curative therapies. Here, we describe a series of pooled, in vivo RNA interference (RNAi) screens to identify essential transcription factors (TFs) in a murine model of acute myeloid leukemia (AML) with genetically- and phenotypically-defined LSCs. These screens reveal the heterodimeric, circadian rhythm TFs Clock and Bmal1 as genes required for the growth of AML cells in vitro and in vivo. Disruption of canonical circadian pathway components produces anti-leukemic effects, including impaired proliferation, enhanced myeloid differentiation, and depletion of LSCs. We find that both normal and malignant hematopoietic cells harbor an intact clock with robust circadian oscillations, and genetic knockout models reveal a leukemia-specific dependence on the pathway. Our findings establish a role for the core circadian clock genes in AML.

SUMMARY We used an in vivo short hairpin RNA (shRNA) screening approach to identify genes that are essential for MLL-AF9 acute myeloid leukemia (AML). We found that Integrin Beta 3 (Itgb3) is essential for murine leukemia cells in vivo, and for human leukemia cells in xenotransplantation studies. In leukemia cells, Itgb3 knockdown impaired homing, downregulated LSC transcriptional programs, and induced differentiation via the intracellular kinase, Syk. In contrast, loss of Itgb3 in normal HSPCs did not affect engraftment, reconstitution, or differentiation. Finally, we confirmed that Itgb3 is dispensable for normal hematopoiesis and required for leukemogenesis using an Itgb3 knockout mouse model. Our results establish the significance of the Itgb3 signaling pathway as a potential therapeutic target in AML.

Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone-marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on-target, via inhibition of HMGCoA reductase. These results illustrate the power of merging physiologically-relevant models with high-throughput screening.

Targeting Csnk1a1 provides a potential therapeutic approach for AML associated with nonmutated Tp53.

Acute myeloid leukemia (AML) is a heterogeneous disease with complex molecular pathophysiology. To systematically characterize AML's genetic dependencies, we conducted genome-scale short hairpin RNA screens in 17 AML cell lines and analyzed dependencies relative to parallel screens in 199 cell lines of other cancer types. We identified 353 genes specifically required for AML cell proliferation. To validate the in vivo relevance of genetic dependencies observed in human cell lines, we performed a secondary screen in a syngeneic murine AML model driven by the MLL-AF9 oncogenic fusion protein. Integrating the results of these interference RNA screens and additional gene expression data, we identified the transcription factor ZEB2 as a novel AML dependency. ZEB2 depletion impaired the proliferation of both human and mouse AML cells and resulted in aberrant differentiation of human AML cells. Mechanistically, we showed that ZEB2 transcriptionally represses genes that regulate myeloid differentiation, including genes involved in cell adhesion and migration. In addition, we found that epigenetic silencing of the miR-200 family microRNAs affects ZEB2 expression. Our results extend the role of ZEB2 beyond regulating epithelial-mesenchymal transition (EMT) and establish ZEB2 as a novel regulator of AML proliferation and differentiation.

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