Among mammals, only eutherians and marsupials are viviparous and have genomic imprinting that leads to parent-of-origin-specific differential gene expression. We used comparative analysis to investigate the origin of genomic imprinting in mammals. PEG10 (paternally expressed 10) is a retrotransposon-derived imprinted gene that has an essential role for the formation of the placenta of the mouse. Here, we show that an orthologue of PEG10 exists in another therian mammal, the marsupial tammar wallaby (Macropus eugenii), but not in a prototherian mammal, the egg-laying platypus (Ornithorhynchus anatinus), suggesting its close relationship to the origin of placentation in therian mammals. We have discovered a hitherto missing link of the imprinting mechanism between eutherians and marsupials because tammar PEG10 is the first example of a differentially methylated region (DMR) associated with genomic imprinting in marsupials. Surprisingly, the marsupial DMR was strictly limited to the 5′ region of PEG10, unlike the eutherian DMR, which covers the promoter regions of both PEG10 and the adjacent imprinted gene SGCE. These results not only demonstrate a common origin of the DMR-associated imprinting mechanism in therian mammals but provide the first demonstration that DMR-associated genomic imprinting in eutherians can originate from the repression of exogenous DNA sequences and/or retrotransposons by DNA methylation.

The outcome of treatment-refractory and/or relapsed pediatric T cell acute lymphoblastic leukemia (T-ALL) is extremely poor, and the genetic basis for this is not well understood. Here we report comprehensive profiling of 121 cases of pediatric T-ALL using transcriptome and/or targeted capture sequencing, through which we identified new recurrent gene fusions involving SPI1 (STMN1-SPI1 and TCF7-SPI1). Cases positive for fusions involving SPI1 (encoding PU.1), accounting for 3.9% (7/181) of the examined pediatric T-ALL cases, showed a double-negative (DN; CD4CD8) or CD8 single-positive (SP) phenotype and had uniformly poor overall survival. These cases represent a subset of pediatric T-ALL distinguishable from the known T-ALL subsets in terms of expression of genes involved in T cell precommitment, establishment of T cell identity, and post-β-selection maturation and with respect to mutational profile. PU.1 fusion proteins retained transcriptional activity and, when constitutively expressed in mouse stem/progenitor cells, induced cell proliferation and resulted in a maturation block. Our findings highlight a unique role of SPI1 fusions in high-risk pediatric T-ALL.

Loss-of-function mutations of EZH2, a catalytic component of polycomb repressive complex 2 (PRC2), are observed in B10% of patients with myelodysplastic syndrome (MDS), but are rare in acute myeloid leukaemia (AML). Recent studies have shown that EZH2 mutations are often associated with RUNX1 mutations in MDS patients, although its pathological function remains to be addressed. Here we establish an MDS mouse model by transducing a RUNX1S291fs mutant into hematopoietic stem cells and subsequently deleting Ezh2. Ezh2 loss significantly promotes RUNX1S291fs-induced MDS. Despite their compromised proliferative capacity of RUNX1S291fs/Ezh2-null MDS cells, MDS bone marrow impairs normal hematopoietic cells via selectively activating inflammatory cytokine responses, thereby allowing propagation of MDS clones. In contrast, loss of Ezh2 prevents the transformation of AML via PRC1-mediated repression of Hoxa9. These findings provide a comprehensive picture of how Ezh2 loss collaborates with RUNX1 mutants in the pathogenesis of MDS in both cell autonomous and non-autonomous manners.

Key Points Ezh2 loss in hematopoietic stem cells predisposes mice to develop heterogeneous hematologic malignancies. Ezh1 is essential to maintain hematopoiesis in the setting of Ezh2 loss.

Loss of Ezh2 in the presence of activating mutation in JAK2 (JAK2V617F) cooperatively alters transcriptional programs of hematopoiesis, activates specific oncogenes, and promotes the development of myelofibrosis.

Responding to various stimuli is indispensable for the maintenance of homeostasis. The downregulation of ribosomal RNA (rRNA) transcription is one of the mechanisms involved in the response to stimuli by various cellular processes, such as cell cycle arrest and apoptosis. Cell differentiation is caused by intra- and extracellular stimuli and is associated with the downregulation of rRNA transcription as well as reduced cell growth. The downregulation of rRNA transcription during differentiation is considered to contribute to reduced cell growth. However, the downregulation of rRNA transcription can induce various cellular processes; therefore, it may positively regulate cell differentiation. To test this possibility, we specifically downregulated rRNA transcription using actinomycin D or a siRNA for Pol I-specific transcription factor IA (TIF-IA) in HL-60 and THP-1 cells, both of which have differentiation potential. The inhibition of rRNA transcription induced cell differentiation in both cell lines, which was demonstrated by the expression of the common differentiation marker CD11b. Furthermore, TIF-IA knockdown in an ex vivo culture of mouse hematopoietic stem cells increased the percentage of myeloid cells and reduced the percentage of immature cells. We also evaluated whether differentiation was induced via the inhibition of cell cycle progression because rRNA transcription is tightly coupled to cell growth. We found that cell cycle arrest without affecting rRNA transcription did not induce differentiation. To the best of our knowledge, our results demonstrate the first time that the downregulation of rRNA levels could be a trigger for the induction of differentiation in mammalian cells. Furthermore, this phenomenon was not simply a reflection of cell cycle arrest. Our results provide a novel insight into the relationship between rRNA transcription and cell differentiation.

Purpose EZH2 and EZH1, the catalytic components of polycomb repressive complex 2 (PRC2), trigger trimethylation of H3K27 (H3K27me3) to repress the transcription of target genes and are implicated in the pathogenesis of various cancers including multiple myeloma and prostate cancer. Here, we investigated the preclinical effects of UNC1999, a dual inhibitor of EZH2 and EZH1, in combination with proteasome inhibitors on multiple myeloma and prostate cancer. Experimental Design In vitro and in vivo efficacy of UNC1999 and the combination with proteasome inhibitors was evaluated in multiple myeloma cell lines, primary patient cells, and in a xenograft model. RNA-seq and ChIP-seq were performed to uncover the targets of UNC1999 in multiple myeloma. The efficacy of the combination therapy was validated in prostate cancer cell lines. Results Proteasome inhibitors repressed EZH2 transcription via abrogation of the RB-E2F pathway, thereby sensitizing EZH2-dependent multiple myeloma cells to EZH1 inhibition by UNC1999. Correspondingly, combination of proteasome inhibitors with UNC1999, but not with an EZH2-specific inhibitor, induced synergistic antimyeloma activity in vitro. Bortezomib combined with UNC1999 remarkably inhibited the growth of myeloma cells in vivo. Comprehensive analyses revealed several direct targets of UNC1999 including the tumor suppressor gene NR4A1. Derepression of NR4A1 by UNC1999 resulted in suppression of MYC, which was enhanced by the combination with bortezomib, suggesting the cooperative blockade of PRC2 function. Notably, this combination also exhibited strong synergy in prostate cancer cells. Conclusions Our results identify dual inhibition of EZH2 and EZH1 together with proteasome inhibition as a promising epigenetics-based therapy for PRC2-dependent cancers.

BackgroundHuman cord blood (hCB) is the main source of hematopoietic stem and progenitor cells (HSCs/PCs) for transplantation. Efforts to overcome relative shortages of HSCs/PCs have led to technologies to expand HSCs/PCs ex vivo. However, methods suitable for clinical practice have yet to be fully established.Methodology/Principal FindingsIn this study, we screened biologically active natural products for activity to promote expansion of hCB HSCs/PCs ex vivo, and identified Garcinol, a plant-derived histone acetyltransferase (HAT) inhibitor, as a novel stimulator of hCB HSC/PC expansion. During a 7-day culture of CD34+CD38– HSCs supplemented with stem cell factor and thrombopoietin, Garcinol increased numbers of CD34+CD38– HSCs/PCs more than 4.5-fold and Isogarcinol, a derivative of Garcinol, 7.4-fold. Furthermore, during a 7-day culture of CD34+ HSCs/PCs, Garcinol expanded the number of SCID-repopulating cells (SRCs) 2.5-fold. We also demonstrated that the capacity of Garcinol and its derivatives to expand HSCs/PCs was closely correlated with their inhibitory effect on HAT. The Garcinol derivatives which expanded HSCs/PCs inhibited the HAT activity and acetylation of histones, while inactive derivatives did not.Conclusions/SignificanceOur findings identify Garcinol as the first natural product acting on HSCs/PCs and suggest the inhibition of HAT to be an alternative approach for manipulating HSCs/PCs.