SUMMARY Stem cell differentiation pathways are most often studied at the population level, whereas critical decisions are executed at the level of single cells. We have established a highly multiplexed, quantitative PCR assay to profile in an unbiased manner a panel of all commonly used cell surface markers (280 genes) from individual cells. With this method we analyzed over 1500 single cells throughout the mouse hematopoietic system, and illustrate its utility for revealing important biological insights. The comprehensive single cell dataset permits mapping of the mouse hematopoietic stem cell (HSC) differentiation hierarchy by computational lineage progression analysis. Further profiling of 180 intracellular regulators enabled construction of a genetic network to assign the earliest differentiation event during hematopoietic lineage specification. Analysis of acute myeloid leukemia elicited by MLL-AF9 uncovered a distinct cellular hierarchy containing two independent self-renewing lineages with different clonal activities. The strategy has broad applicability in other cellular systems.

Enhancer of zeste homolog2 (EZH2) is the histone lysine N-methyltransferase component of the Polycomb repressive complex 2 (PRC2), which in conjunction with embryonic ectoderm development (EED) and suppressor of zeste 12 homolog (SUZ12), regulates cell lineage determination and homeostasis. Enzymatic hyperactivity has been linked to aberrant repression of tumor suppressor genes in diverse cancers. Here, we report the development of stabilized alpha-helix of EZH2 (SAH-EZH2) peptides that selectively inhibit H3 Lys27 trimethylation by dose-responsively disrupting the EZH2/EED complex and reducing EZH2 protein levels, a mechanism distinct from that reported for small molecule EZH2 inhibitors targeting the enzyme catalytic domain. MLL-AF9 leukemia cells, which are dependent on PRC2, undergo growth arrest and monocyte/macrophage differentiation upon treatment with SAH-EZH2, consistent with observed changes in expression of PRC2-regulated, lineage-specific marker genes. Thus, by dissociating the EZH2/EED complex, we pharmacologically modulate an epigenetic “writer” and suppress PRC2-dependent cancer cell growth.

A growing body of data suggests the importance of epigenetic mechanisms in cancer. Polycomb repressive complex 2 (PRC2) has been implicated in self-renewal and cancer progression, and its components are overexpressed in many cancers. However, its role in cancer development and progression remains unclear. We used conditional alleles for the PRC2 components enhancer of zeste 2 (Ezh2) and embryonic ectoderm development (Eed) to characterize the role of PRC2 function in leukemia development and progression. Compared with wild-type leukemia, Ezh2-null MLL-AF9-mediated acute myeloid leukemia (AML) failed to accelerate upon secondary transplantation. However, Ezh2-null leukemias maintained self-renewal up to the third round of transplantation, indicating that Ezh2 is not strictly required for MLL-AF9 AML, but plays a role in leukemia progression. Genome-wide analyses of PRC2-mediated trimethylation of histone 3 demonstrated locus-specific persistence of H3K27me3 despite inactivation of Ezh2, suggesting partial compensation by Ezh1. In contrast, inactivation of the essential PRC2 gene, Eed, led to complete ablation of PRC2 function, which was incompatible with leukemia growth. Gene expression array analyses indicated more profound gene expression changes in Eed-null compared with Ezh2-null leukemic cells, including down-regulation of Myc target genes and up-regulation of PRC2 targets. Manipulating PRC2 function may be of therapeutic benefit in AML. epigenetics | mouse model | polycomb group proteins | myeloid-lymphoid leukemia protein P olycomb repressive complex 2 (PRC2) has been implicated in development and cancer (1, 2). PRC2 is composed of the core components embryonic ectoderm development (EED), suppressor of zeste 12 (SUZ12), and a SET-domain methyltransferase, either enhancer of zeste 2 (EZH2) or enhancer of zeste 1 (EZH1) (3). The PRC2 complex catalyzes the di-and trimethylation of lysine residue 27 of histone 3 (H3K27me3), a repressive chromatin mark (4). Overexpression of EZH2 has been correlated with prostate cancer progression (5). Follow-up studies have confirmed and expanded these results for other cancer types, mainly solid tumors. In the hematopoietic system, forced expression of Ezh2 increases serial transplantation potential in hematopoietic stem cells (6) and enhances transformation in a model of multiple myeloma (7). Intriguingly, heterozygous loss of function of EZH2 has been associated with adverse prognosis in myelofibrosis (8), and heterozygous and homozygous inactivation of EZH2 has been described in myelodysplastic syndromes and more recently in Tlineage lymphoblastic leukemia (9-12). In contrast, EZH2 alterations appear to be rare events in acute myeloid leukemia (AML). How to reconcile these findings on a mechanistic level is unclear. Given the heterogeneity of clinical samples, elucidating the complex role of PRC2 biology in cancer will be aided by studies in genetically defined animal models (13).Loss of function of PRC2 has been evaluated in several cancer models (14, 15). However, shRNA...

No abstract