Summary The histone 3 lysine 79 (H3K79) methyltransferase Dot1l has been implicated in the development of leukemias bearing translocations of the Mixed Lineage Leukemia (MLL) gene. We identified the MLL-fusion targets in an MLL-AF9 leukemia model, and conducted epigenetic profiling for H3K79me2, H3K4me3, H3K27me3 and H3K36me3 in hematopoietic progenitor and leukemia stem cells (LSC). We found abnormal profiles only for H3K79me2 on MLL-AF9 fusion target loci in LSC. Inactivation of Dot1l lead to down-regulation of direct MLL-AF9 targets and an MLL-translocation associated gene expression signature, while global gene expression remained largely unaffected. Suppression of MLL-translocation associated gene expression corresponded with dependence of MLL-AF9 leukemia on Dot1l in vivo. These data point to DOT1L as a potential therapeutic target in MLL-rearranged leukemia.

Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodeling1. While several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming2,3, the role of specific chromatin modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used shRNAs to target genes in DNA and histone methylation pathways, and have identified positive and negative modulators of iPSC generation. While inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase Ezh2, reduced reprogramming efficiency, suppression of SUV39H1, YY1, and Dot1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase Dot1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for Klf4 and c-Myc. Inhibition of Dot1L early in the reprogramming process is associated with a marked increase in two alternative factors, Nanog and Lin28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. Dot1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors.

In recent years, long noncoding RNAs (lncRNAs) have been shown to have critical regulatory roles in cancer biology. However, the contributions of lncRNAs to hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) remain largely unknown. Differentially expressed lncRNAs between HBV-related HCC and paired peritumoral tissues were identified by microarray and validated using quantitative real-time polymerase chain reaction. Liver samples from patients with HBV-related HCC were analyzed for levels of a specific differentially expressed lncRNA High Expression In HCC (termed lncRNA-HEIH); data were compared with survival data using the Kaplan-Meier method and compared between groups by the log-rank test. The effects of lncRNA-HEIH were assessed by silencing and overexpressing the lncRNA in vitro and in vivo. The expression level of lncRNA-HEIH in HBV-related HCC is significantly associated with recurrence and is an independent prognostic factor for survival. We also found that lncRNA-HEIH plays a key role in G 0 /G 1 arrest, and further demonstrated that lncRNA-HEIH was associated with enhancer of zeste homolog 2 (EZH2) and that this association was required for the repression of EZH2 target genes. Conclusions: Together, these results indicate that lncRNA-HEIH is an oncogenic lncRNA that promotes tumor progression and leads us to propose that lncRNAs may serve as key regulatory hubs in HCC progression. (HEPATOLOGY 2011;54:1679-1689 H epatocellular carcinoma (HCC) is one of the most common human cancers worldwide, particularly in Southeast Asia and Africa. 1More than 70%-80% of HCC cases occur in high hepatitis B virus (HBV) endemic regions, and 50% of HCC cases worldwide are attributable to chronic infection with HBV. Unfortunately, the 5-year survival rate of HBV-related HCC patients remains poor, and approximately 600,000 HCC patients die each year, despite recent advances in surgical techniques and medical treatment.2 Although previous studies identified many aberrantly expressed protein-coding genes in HCC, novel molecular markers that can help in early diagnosis and risk assessment are still urgently needed. 3It is of paramount importance to understand the relationships between clinical symptoms and molecular changes in HCC for developing new diagnosis and treatment strategies for HCC and improving the prognosis of diagnosed patients. The human transcriptome comprises not only large numbers of protein-coding messenger RNAs (mRNAs), but also a large set of nonprotein coding Abbreviations:: CCK-8, Cell-Counting Kit-8 assay; ChIP, chromatin immunoprecipitation; cDNA, complementary DNA; 95% CI, 95% confidence interval; EZH2, enhancer of zeste homolog

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...

MLL -rearrangements generate MLL-fusion proteins that bind DNA and drive leukemogenic gene expression. This gene expression program is dependent on the histone 3 lysine 79 (H3K79) methyltransferase DOT1L, and small molecule DOT1L inhibitors show promise as therapeutics for these leukemias. However, the mechanisms underlying this dependency are unclear. We conducted a genome-scale RNAi screen and found that the histone deacetylase SIRT1 is required for the establishment of a heterochromatin-like state around MLL-fusion target genes after DOT1L inhibition. DOT1L inhibits chromatin localization of a repressive complex composed of SIRT1 and SUV39H1, thereby maintaining an open chromatin state with elevated H3K9 acetylation and minimal H3K9 methylation at MLL-fusion target genes. Furthermore, the combination of SIRT1 activators and DOT1L inhibitors shows enhanced activity against MLL-rearranged leukemia cells. These results indicate that the dynamic interplay between chromatin regulators controlling activation and repression of gene expression could provide novel opportunities for combination therapy.

One-year survival rates for newly diagnosed hepatocellular carcinoma (HCC) are <50%, and unresectable HCC carries a dismal prognosis owing to its aggressiveness and the undruggable nature of its main genetic drivers. By screening a custom library of shRNAs directed toward known drug targets in a genetically defined Myc-driven HCC model, we identified cyclin-dependent kinase 9 (Cdk9) as required for disease maintenance. Pharmacological or shRNA-mediated CDK9 inhibition led to robust anti-tumor effects that correlated with MYC expression levels and depended on the role that both CDK9 and MYC exert in transcription elongation. Our results establish CDK9 inhibition as a therapeutic strategy for MYC-overexpressing liver tumors and highlight the relevance of transcription elongation in the addiction of cancer cells to MYC.

SUMMARY Homeotic (HOX) genes are dysregulated in multiple malignancies including several AML subtypes. We demonstrate that H3K79 dimethylation (H3K79me2) is converted to mono-methylation (H3K79me1) at HOX loci as hematopoietic cells mature thus coinciding with a decrease in HOX gene expression. We show that H3K79 methyltransferase activity as well as H3K79me1 to H3K79me2 conversion is regulated by the DOT1L co-factor AF10. AF10 inactivation reverses leukemia-associated epigenetic profiles, precludes abnormal HOXA gene expression and impairs the transforming ability of MLL-AF9, MLL-AF6 or NUP98-NSD1 fusions – mechanistically distinct HOX-activating oncogenes. Furthermore, NUP98-NSD1 transformed cells are sensitive to small-molecule inhibition of DOT1L. Our findings demonstrate that pharmacological inhibition of the DOT1L/AF10 complex may provide therapeutic benefit in an array of malignancies with abnormal HOXA gene expression.

Notch behaves as a tumor suppressor in AML, and Notch activation induces cell cycle arrest, differentiation, and apoptosis of AML-initiating cells.