Person:

Orkin, Stuart

A relatively small cadre of lineage-restricted transcription factors largely orchestrates erythropoiesis, but how these nuclear factors interact to regulate this complex biology is still largely unknown. However, recent technological advances, such as chromatin immunoprecipitation (ChIP) paired with massively parallel sequencing (ChIP-seq), gene expression profiling, and comprehensive bioinformatic analyses, offer new insights into the intricacies of red cell molecular circuits.

Tumors are thought to be sustained by a reservoir of self-renewing cells, termed tumor initiating cells or cancer stem cells. Osteosarcomas are high-grade sarcomas derived from osteoblast progenitor cells and are the most common pediatric bone malignancy. In this report we show that the stem cell transcription factor Sox2 is highly expressed in human and murine osteosarcoma cell lines as well as in tumor samples. Osteosarcoma cells have increased ability to grow in suspension as osteospheres, that are greatly enriched in expression of Sox2 and the stem cell marker, Sca-1. Depletion of Sox2 by shRNAs in independent murine osteosarcoma-derived cells drastically reduces their transformed properties in vitro and their ability to form tumors. Sox2-depleted osteosarcoma cells can no longer form osteospheres, and differentiate into mature osteoblasts. Concomitantly, they exhibit decreased Sca-1 expression and upregulation of the Wnt signaling pathway. Thus, despite other mutations, these tumor cells maintain a proliferative requirement for Sox2. Our data indicate that Sox2 is required for osteosarcoma cell self-renewal, and that Sox2 antagonizes the pro-differentiation Wnt pathway, that can in turn reduce Sox2 expression. These studies define Sox2 as a survival factor and a novel biomarker of self-renewal in osteosarcomas, and support a tumor suppressive role for the Wnt pathway in tumors of mesenchymal origin. Our findings could provide the basis for novel therapeutic strategies based on inhibiting Sox2 or enhancing Wnt signaling for the treatment of osteosarcomas.

Embryonic stem (ES) cells are of great interest as a model system for studying early developmental processes and because of their potential therapeutic applications in regenerative medicine. Obtaining a systematic understanding of the mechanisms that control the 'stemness' - self-renewal and pluripotency - of ES cells relies on high-throughput tools to define gene expression and regulatory networks at the genome level. Such recently developed systems biology approaches have revealed highly interconnected networks in which multiple regulatory factors act in combination. Interestingly, stem cells and cancer cells share some properties, notably self-renewal and a block in differentiation. Recently, several groups reported that expression signatures that are specific to ES cells are also found in many human cancers and in mouse cancer models, suggesting that these shared features might inform new approaches for cancer therapy. Here, we briefly summarize the key transcriptional regulators that contribute to the pluripotency of ES cells, the factors that account for the common gene expression patterns of ES and cancer cells, and the implications of these observations for future clinical applications.

The retinoblastoma protein, Rb, was one of the first tumor suppressor genes identified as a result of the familial syndrome retinoblastoma. In the period since its identification and cloning a large number of studies have described its role in various cellular processes. The application of conditional somatic mutation with lineage and temporally controlled gene deletion strategies, thus circumventing the lethality associated with germ-line deletion of Rb, have allowed for a reanalysis of the in vivo role of Rb. In the hematopoietic system, such approaches have led to new insights into stem cell biology and the role of the microenvironment in regulating hematopoietic stem cell fate. They have also clarified the role that Rb plays during erythropoiesis and defined a novel mechanism linking mitochondrial function to terminal cell cycle withdrawal. These studies have shed light on the in vivo role of Rb in the regulation of hematopoiesis and also prompt further analysis of the role that Rb plays in both the regulation of hematopoietic stem cells and the terminal differentiation of their progeny.

RNA interference (RNAi) is a powerful strategy for studying the phenotypic consequences of reduced gene expression levels in model systems. To develop a method for the rapid characterization of the developmental consequences of gene dysregulation, we tested the use of RNAi for “transient transgenic” knockdown of mRNA in mouse embryos. These methods included lentiviral infection as well as transposition using the Sleeping Beauty (SB) and PiggyBac (PB) transposable element systems. This approach can be useful for phenotypic validation of putative mutant loci, as we demonstrate by confirming that knockdown of Prdm16 phenocopies the ENU-induced cleft palate (CP) mutant, csp1. This strategy is attractive as an alternative to gene targeting in embryonic stem cells, as it is simple and yields phenotypic information in a matter of weeks. Of the three methodologies tested, the PB transposon system produced high numbers of transgenic embryos with the expected phenotype, demonstrating its utility as a screening method.

In development, lineage-restricted transcription factors simultaneously promote differentiation while repressing alternative fates. Molecular dissection of this process has been challenging as transcription factor loci are regulated by many trans-acting factors functioning through dispersed cis elements. It is not understood whether these elements function collectively to confer transcriptional regulation, or individually to control specific aspects of activation or repression, such as initiation versus maintenance. Here, we have analyzed cis element regulation of the critical hematopoietic factor Gata2, which is expressed in early precursors and repressed as GATA-1 levels rise during terminal differentiation. We engineered mice lacking a single cis element −1.8 kb upstream of the Gata2 transcriptional start site. Although Gata2 is normally repressed in late-stage erythroblasts, the −1.8 kb mutation unexpectedly resulted in reactivated Gata2 transcription, blocked differentiation, and an aberrant lineage-specific gene expression pattern. Our findings demonstrate that the −1.8 kb site selectively maintains repression, confers a specific histone modification pattern and expels RNA Polymerase II from the locus. These studies reveal how an individual cis element establishes a normal developmental program via regulating specific steps in the mechanism by which a critical transcription factor is repressed.

In proliferating cells, where most Polycomb repressive complex 2 (PRC2) studies have been performed, gene repression is associated with PRC2 trimethylation of H3K27 (H3K27me3). However, it is uncertain whether PRC2 writing of H3K27me3 is mechanistically required for gene silencing. Here, we studied PRC2 function in postnatal mouse cardiomyocytes, where the paucity of cell division obviates bulk H3K27me3 rewriting after each cell cycle. EED (embryonic ectoderm development) inactivation in the postnatal heart (EedCKO) caused lethal dilated cardiomyopathy. Surprisingly, gene upregulation in EedCKO was not coupled with loss of H3K27me3. Rather, the activating histone mark H3K27ac increased. EED interacted with histone deacetylases (HDACs) and enhanced their catalytic activity. HDAC overexpression normalized EedCKO heart function and expression of derepressed genes. Our results uncovered a non-canonical, H3K27me3-independent EED repressive mechanism that is essential for normal heart function. Our results further illustrate that organ dysfunction due to epigenetic dysregulation can be corrected by epigenetic rewiring. DOI: http://dx.doi.org/10.7554/eLife.24570.001

Histone deacetylase (HDAC)-inhibitors (HDACis) are well characterized anti-cancer agents with promising results in clinical trials. However, mechanistically little is known regarding their selectivity in killing malignant cells while sparing normal cells. Gene expression-based chemical genomics identified HDACis as being particularly potent against Down syndrome associated myeloid leukemia (DS-AMKL) blasts. Investigating the anti-leukemic function of HDACis revealed their transcriptional and posttranslational regulation of key autophagic proteins, including ATG7. This leads to suppression of autophagy, a lysosomal degradation process that can protect cells against damaged or unnecessary organelles and protein aggregates. DS-AMKL cells exhibit low baseline autophagy due to mTOR activation. Consequently, HDAC inhibition repressed autophagy below a critical threshold, which resulted in accumulation of mitochondria, production of reactive oxygen species, DNA-damage and apoptosis. Those HDACi-mediated effects could be reverted upon autophagy activation or aggravated upon further pharmacological or genetic inhibition. Our findings were further extended to other major acute myeloid leukemia subgroups with low basal level autophagy. The constitutive suppression of autophagy due to mTOR activation represents an inherent difference between cancer and normal cells. Thus, via autophagy suppression, HDACis deprive cells of an essential pro-survival mechanism, which translates into an attractive strategy to specifically target cancer cells.

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.

Transient leukemia (TL) is evident in 5–10% of all neonates with Down syndrome (DS) and associated with N-terminal truncating GATA1-mutations (GATA1s). Here we report that TL cell clones generate abundant eosinophils in a substantial fraction of patients. Sorted eosinophils from patients with TL and eosinophilia carried the same GATA1s-mutation as sorted TL-blasts, consistent with their clonal origin. TL-blasts exhibited a genetic program characteristic of eosinophils and differentiated along the eosinophil lineage in vitro. Similarly, ectopic expression of Gata1s, but not Gata1, in wild-type CD34+-hematopoietic stem and progenitor cells induced hyperproliferation of eosinophil promyelocytes in vitro. While GATA1s retained the function of GATA1 to induce eosinophil genes by occupying their promoter regions, GATA1s was impaired in its ability to repress oncogenic MYC and the pro-proliferative E2F transcription network. ChIP-seq indicated reduced GATA1s occupancy at the MYC promoter. Knockdown of MYC, or the obligate E2F-cooperation partner DP1, rescued the GATA1s-induced hyperproliferative phenotype. In agreement, terminal eosinophil maturation was blocked in Gata1Δe2 knockin mice, exclusively expressing Gata1s, leading to accumulation of eosinophil precursors in blood and bone marrow. These data suggest a direct relationship between the N-terminal truncating mutations of GATA1 and clonal eosinophilia in DS patients.