Person:

Guo, Guoji

Background: A fundamental challenge for cancer therapy is that each tumor contains a highly heterogeneous cell population whose structure and mechanistic underpinnings remain incompletely understood. Recent advances in single-cell gene expression profiling have created new possibilities to characterize this heterogeneity and to dissect the potential intra-cancer cellular hierarchy. Results: Here, we apply single-cell analysis to systematically characterize the heterogeneity within leukemic cells using the MLL-AF9 driven mouse model of acute myeloid leukemia. We start with fluorescence-activated cell sorting analysis with seven surface markers, and extend by using a multiplexing quantitative polymerase chain reaction approach to assay the transcriptional profile of a panel of 175 carefully selected genes in leukemic cells at the single-cell level. By employing a set of computational tools we find striking heterogeneity within leukemic cells. Mapping to the normal hematopoietic cellular hierarchy identifies two distinct subtypes of leukemic cells; one similar to granulocyte/monocyte progenitors and the other to macrophage and dendritic cells. Further functional experiments suggest that these subtypes differ in proliferation rates and clonal phenotypes. Finally, co-expression network analysis reveals similarities as well as organizational differences between leukemia and normal granulocyte/monocyte progenitor networks. Conclusions: Overall, our single-cell analysis pinpoints previously uncharacterized heterogeneity within leukemic cells and provides new insights into the molecular signatures of acute myeloid leukemia. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0525-9) contains supplementary material, which is available to authorized users.

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.

Here, we describe that lysine-specific demethylase 1 (Lsd1/KDM1a), which demethylates histone H3 on Lys4 or Lys9 (H3K4/K9), is an indispensible epigenetic governor of hematopoietic differentiation. Integrative genomic analysis, combining global occupancy of Lsd1, genome-wide analysis of its substrates H3K4 monomethylation and dimethylation, and gene expression profiling, reveals that Lsd1 represses hematopoietic stem and progenitor cell (HSPC) gene expression programs during hematopoietic differentiation. We found that Lsd1 acts at transcription start sites, as well as enhancer regions. Loss of Lsd1 was associated with increased H3K4me1 and H3K4me2 methylation on HSPC genes and gene derepression. Failure to fully silence HSPC genes compromised differentiation of hematopoietic stem cells as well as mature blood cell lineages. Collectively, our data indicate that Lsd1-mediated concurrent repression of enhancer and promoter activity of stem and progenitor cell genes is a pivotal epigenetic mechanism required for proper hematopoietic maturation. DOI: http://dx.doi.org/10.7554/eLife.00633.001

The biology of hematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions. Particularly challenging has been the study of dynamic HSC behaviors in the native niche given that live animal HSC tracking under steady state conditions still represents an elusive goal in the field. Here, we describe a dual genetic strategy in mice that restricts reporter labeling to a subset of the most quiescent longterm HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial marrow. We find that this subset of LT-HSCs resides in an endostealsinusoidal niche where they are simultaneously in close proximity to sinusoidal blood vessels and the endosteal surface. In contrast, multipotent progenitor cells (MPPs) display a broader distance distribution from the endosteum and are more likely to be associated with transition zone vessels. Additionally, our results demonstrate that LTHSCs do not occupy the marrow niches with the deepest hypoxia and that they are found in similar hypoxic environments as MPPs. In vivo time-lapse imaging experiments revealed that LT-HSCs display limited motility compared to the more motile MPPs. However, following activation, LT-HSCs become more motile and expand clonally within spatially restricted domains. These spatial domains have defined characteristics, as HSC expansion is found almost exclusively in a subset of bone marrow cavities exhibiting bone-remodeling activities (resorption and new bone deposition). In contrast, cavities with low bone-resorbing activities do not harbor expanding HSCs. These findings point to a new degree of heterogeneity within the bone marrow microenvironment, imposed by the stages of bone turnover, which has not been recognized previously. Overall, our work describes a model that enables live imaging of LT-HSCs in the native niche and provides insight into the dynamic behaviors of hematopoietic stem and progenitor cells, and the heterogeneity of HSC niches.