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DNA hypomethylating agents (HMAs) are used for the treatment of myeloid malignancies, although their therapeutic effects have been unsatisfactory. Here we show that CRISPR-Cas9 screening reveals that knockout of topoisomerase 1-binding arginine/serine-rich protein (TOPORS), which encodes a ubiquitin/SUMO E3 ligase, augments the efficacy of HMAs on myeloid leukemic cells with little effect on normal hematopoiesis, suggesting that TOPORS is involved in resistance to HMAs. HMAs are incorporated into the DNA and trap DNA methyltransferase-1 (DNMT1) to form DNA-DNMT1 crosslinks, which undergo SUMOylation, followed by proteasomal degradation. Persistent crosslinking is cytotoxic. The TOPORS RING finger domain, which mediates ubiquitination, is responsible for HMA resistance. In TOPORS knockout cells, DNMT1 is stabilized by HMA treatment due to inefficient ubiquitination, resulting in the accumulation of unresolved SUMOylated DNMT1. This indicates that TOPORS ubiquitinates SUMOylated DNMT1, thereby promoting the resolution of DNA-DNMT1 crosslinks. Consistently, the ubiquitination inhibitor, TAK-243, and the SUMOylation inhibitor, TAK-981, show synergistic effects with HMAs through DNMT1 stabilization. Our study provides a novel HMA-based therapeutic strategy that interferes with the resolution of DNA-DNMT1 crosslinks., (© 2024. The Author(s).)

Poor survival and lack of treatment response in glioblastoma (GBM) is attributed to the persistence of glioma stem cells (GSCs). To identify novel therapeutic approaches, we performed CRISPR/Cas9 knockout screens and discovered TGFβ activated kinase (TAK1) as a selective survival factor in a significant fraction of GSCs. Loss of TAK1 kinase activity results in RIPK1-dependent apoptosis via Caspase-8/FADD complex activation, dependent on autocrine TNFα ligand production and constitutive TNFR signaling. We identify a transcriptional signature associated with immune activation and the mesenchymal GBM subtype to be a characteristic of cancer cells sensitive to TAK1 perturbation and employ this signature to accurately predict sensitivity to the TAK1 kinase inhibitor HS-276. In addition, exposure to pro-inflammatory cytokines IFNγ and TNFα can sensitize resistant GSCs to TAK1 inhibition. Our findings reveal dependency on TAK1 kinase activity as a novel vulnerability in immune-activated cancers, including mesenchymal GBMs that can be exploited therapeutically., (© 2024. The Author(s).)

The human silencing hub (HUSH) complex is an epigenetic repressor complex whose role has emerged as an important guardian of genome integrity. It protects the genome from exogenous DNA invasion and regulates endogenous retroelements by recruiting histone methyltransferases catalyzing histone 3 lysine 9 trimethylation (H3K9me3) and additional proteins involved in chromatin compaction. In particular, its regulation of transcriptionally active LINE1 retroelements, by binding to and neutralizing LINE1 transcripts, has been well characterized. HUSH is required for mouse embryogenesis and is associated with disease, in particular cancer. Here we provide insights into the structural and biochemical features of the HUSH complex. Furthermore, we discuss the molecular mechanisms by which the HUSH complex is recruited to specific genomic regions and how it silences transcription. Finally, we discuss the role of HUSH complex members in mammalian development, antiretroviral immunity, and diseases such as cancer., (© 2024. Springer Nature America, Inc.)

Mutations in the epigenetic modifier TET2 are frequent in myeloid malignancies and clonal hematopoiesis of indeterminate potential (CHIP) and clonal cytopenia of undetermined significance (CCUS). Here, we investigate associations between TET2 mutations and DNA methylation in whole blood in 305 elderly twins, 15 patients with CCUS and 18 healthy controls. We find that TET2 mutations are associated with DNA hypermethylation at enhancer sites in whole blood in CHIP and in both granulocytes and mononuclear cells in CCUS. These hypermethylated sites are associated with leukocyte function and immune response and ETS-related and C/EBP-related transcription factor motifs. While the majority of TET2-associated hypermethylation sites are shared between CHIP and in AML, we find a set of AML-specific hypermethylated loci at active enhancer elements in hematopoietic stem cells. In summary, we show that TET2 mutations is associated with hypermethylated enhancers involved in myeloid differentiation in both CHIP, CCUS and AML patients., (© 2021. The Author(s).)

Deciphering the mechanisms that control the pluripotent ground state is key for understanding embryonic development. Nonetheless, the epigenetic regulation of ground-state mouse embryonic stem cells (mESCs) is not fully understood. Here, we identify the epigenetic protein MPP8 as being essential for ground-state pluripotency. Its depletion leads to cell cycle arrest and spontaneous differentiation. MPP8 has been suggested to repress LINE1 elements by recruiting the human silencing hub (HUSH) complex to H3K9me3-rich regions. Unexpectedly, we find that LINE1 elements are efficiently repressed by MPP8 lacking the chromodomain, while the unannotated C-terminus is essential for its function. Moreover, we show that SETDB1 recruits MPP8 to its genomic target loci, whereas transcriptional repression of LINE1 elements is maintained without retaining H3K9me3 levels. Taken together, our findings demonstrate that MPP8 protects the DNA-hypomethylated pluripotent ground state through its association with the HUSH core complex, however, independently of detectable chromatin binding and maintenance of H3K9me3.

Sclerosing stromal tumor (SST) of the ovary is a rare type of sex cord-stromal tumor (SCST), whose genetic underpinning is currently unknown. Here, using whole-exome, targeted capture and RNA-sequencing, we report recurrent FHL2-GLI2 fusion genes in 65% (17/26) of SSTs and other GLI2 rearrangements in additional 15% (4/26) SSTs, none of which are detected in other types of SCSTs (n = 48) or common cancer types (n = 9,950). The FHL2-GLI2 fusions result in transcriptomic activation of the Sonic Hedgehog (SHH) pathway in SSTs. Expression of the FHL2-GLI2 fusion in vitro leads to the acquisition of phenotypic characteristics of SSTs, increased proliferation, migration and colony formation, and SHH pathway activation. Targeted inhibition of the SHH pathway results in reversal of these oncogenic properties, indicating its role in the pathogenesis of SSTs. Our results demonstrate that the FHL2-GLI2 fusion is likely the oncogenic driver of SSTs, defining a genotypic-phenotypic correlation in ovarian neoplasms.

Protein arginine methyltransferase 5 (PRMT5) has emerged as a promising cancer drug target, and three PRMT5 inhibitors are currently in clinical trials for multiple malignancies. In this study, we investigated the role of PRMT5 in human acute myeloid leukemia (AML). Using an enzymatic dead version of PRMT5 and a PRMT5-specific inhibitor, we demonstrated the requirement of the catalytic activity of PRMT5 for the survival of AML cells. We then identified PRMT5 substrates using multiplexed quantitative proteomics and investigated their role in the survival of AML cells. We found that the function of the splicing regulator SRSF1 relies on its methylation by PRMT5 and that loss of PRMT5 leads to changes in alternative splicing of multiple essential genes. Our study proposes a mechanism for the requirement of PRMT5 for leukemia cell survival and provides potential biomarkers for the treatment response to PRMT5 inhibitors.

The chronic skin inflammation psoriasis is crucially dependent on the IL-23/IL-17 cytokine axis. Although IL-23 is expressed by psoriatic keratinocytes and immune cells, only the immune cell-derived IL-23 is believed to be disease relevant. Here we use a genetic mouse model to show that keratinocyte-produced IL-23 is sufficient to cause a chronic skin inflammation with an IL-17 profile. Furthermore, we reveal a cell-autonomous nuclear function for the actin polymerizing molecule N-WASP, which controls IL-23 expression in keratinocytes by regulating the degradation of the histone methyltransferases G9a and GLP, and H3K9 dimethylation of the IL-23 promoter. This mechanism mediates the induction of IL-23 by TNF, a known inducer of IL-23 in psoriasis. Finally, in keratinocytes of psoriatic lesions a decrease in H3K9 dimethylation correlates with increased IL-23 expression, suggesting relevance for disease. Taken together, our data describe a molecular pathway where epigenetic regulation of keratinocytes can contribute to chronic skin inflammation.

Polycomb repressive complex 2 (PRC2) catalyzes methylation on lysine 27 of histone H3 (H3K27) and is required for maintaining transcriptional patterns and cellular identity, but the specification and maintenance of genomic PRC2 binding and H3K27 methylation patterns remain incompletely understood. Epigenetic mechanisms have been proposed, wherein pre-existing H3K27 methylation directs recruitment and regulates the catalytic activity of PRC2 to support its own maintenance. Here we investigate whether such mechanisms are required for specifying H3K27 methylation patterns in mouse embryonic stem cells (mESCs). Through re-expression of PRC2 subunits in PRC2-knockout cells that have lost all H3K27 methylation, we demonstrate that methylation patterns can be accurately established de novo. We find that regional methylation kinetics correlate with original methylation patterns even in their absence, and specification of the genomic PRC2 binding pattern is retained and specifically dependent on the PRC2 core subunit SUZ12. Thus, the H3K27 methylation patterns in mESCs are not dependent on self-autonomous epigenetic inheritance.

Enhancer of zeste homologue 2 (EZH2), the catalytic subunit of Polycomb repressive complex 2 (PRC2), has attracted broad research attention in the past few years because of its involvement in the development and maintenance of many types of cancer and the use of specific EZH2 inhibitors in clinical trials. Several observations show that PRC2 can have both oncogenic and tumour-suppressive functions. We propose that these apparently opposing roles of PRC2 in cancer are a consequence of the molecular function of the complex in maintaining, rather than specifying, the transcriptional repression state of its several thousand target genes.

The Polycomb repressive complexes PRC1 and PRC2 play a central role in developmental gene regulation in multicellular organisms. PRC1 and PRC2 modify chromatin by catalysing histone H2A lysine 119 ubiquitylation (H2AK119u1), and H3 lysine 27 methylation (H3K27me3), respectively. Reciprocal crosstalk between these modifications is critical for the formation of stable Polycomb domains at target gene loci. While the molecular mechanism for recognition of H3K27me3 by PRC1 is well defined, the interaction of PRC2 with H2AK119u1 is poorly understood. Here we demonstrate a critical role for the PRC2 cofactor Jarid2 in mediating the interaction of PRC2 with H2AK119u1. We identify a ubiquitin interaction motif at the amino-terminus of Jarid2, and demonstrate that this domain facilitates PRC2 localization to H2AK119u1 both in vivo and in vitro. Our findings ascribe a critical function to Jarid2 and define a key mechanism that links PRC1 and PRC2 in the establishment of Polycomb domains.

Oncogene-induced senescence (OIS) can occur in response to oncogenic insults and is considered an important tumour suppressor mechanism. Here we identify the lncRNA MIR31HG as upregulated in OIS and find that knockdown of MIR31HG promotes a strong p16(INK4A)-dependent senescence phenotype. Under normal conditions, MIR31HG is found in both nucleus and cytoplasm, but following B-RAF expression MIR31HG is located mainly in the cytoplasm. We show that MIR31HG interacts with both INK4A and MIR31HG genomic regions and with Polycomb group (PcG) proteins, and that MIR31HG is required for PcG-mediated repression of the INK4A locus. We further identify a functional enhancer, located between MIR31HG and INK4A, which becomes activated during OIS and interacts with the MIR31HG promoter. Data from melanoma patients show a negative correlation between MIR31HG and p16(INK4A) expression levels, suggesting a role for this transcript in cancer. Hence, our data provide a new lncRNA-mediated regulatory mechanism for the tumour suppressor p16(INK4A).

The ability of PRC1 and PRC2 to promote proliferation is a main feature that links polycomb (PcG) activity to cancer. PcGs silence the expression of the tumour suppressor locus Ink4a/Arf, whose products positively regulate pRb and p53 functions. Enhanced PcG activity is a frequent feature of human tumours, and PcG inhibition has been proposed as a strategy for cancer treatment. However, the recurrent inactivation of pRb/p53 responses in human cancers raises a question regarding the ability of PcG proteins to affect cellular proliferation independently from this checkpoint. Here we demonstrate that PRCs regulate cellular proliferation and transformation independently of the Ink4a/Arf-pRb-p53 pathway. We provide evidence that PRCs localize at replication forks, and that loss of their function directly affects the progression and symmetry of DNA replication forks. Thus, we have identified a novel activity by which PcGs can regulate cell proliferation independently of major cell cycle restriction checkpoints.

It has recently been demonstrated that the genes controlling the epigenetic programmes that are required for maintaining chromatin structure and cell identity include genes that drive human cancer. This observation has led to an increased awareness of chromatin-associated proteins as potentially interesting drug targets. The successful introduction of DNA methylation and histone deacetylase (HDAC) inhibitors for the treatment of specific subtypes of cancer has paved the way for the use of epigenetic therapy. Here, we highlight key biological findings demonstrating the roles of members of the histone lysine demethylase class of enzymes in the development of cancers, discuss the potential and challenges of therapeutically targeting them, and highlight emerging small-molecule inhibitors of these enzymes.

Polycomb group (PcG) proteins are epigenetic regulators of transcription that have key roles in stem-cell identity, differentiation and disease. Mechanistically, they function within multiprotein complexes, called Polycomb repressive complexes (PRCs), which modify histones (and other proteins) and silence target genes. The dynamics of PRC1 and PRC2 components has been the focus of recent research. Here we discuss our current knowledge of the PRC complexes, how they are targeted to chromatin and how the high diversity of the PcG proteins allows these complexes to influence cell identity.

Histone modifications are thought to regulate chromatin structure, transcription and other nuclear processes. Histone methylation was originally believed to be an irreversible modification that could only be removed by histone eviction or by dilution during DNA replication. However, the isolation of two families of enzymes that can demethylate histones has changed this notion. The biochemical activities of these histone demethylases towards specific Lys residues on histones, and in some cases non-histone substrates, have highlighted their importance in developmental control, cell-fate decisions and disease. Their ability to be regulated through protein-targeting complexes and post-translational modifications is also beginning to shed light on how they provide dynamic control during transcription.

The Polycomb group (PcG) proteins are transcriptional repressors that regulate lineage choices during development and differentiation. Recent studies have advanced our understanding of how the PcG proteins regulate cell fate decisions and how their deregulation potentially contributes to cancer. In this Review we discuss the emerging roles of long non-coding RNAs (ncRNAs) and a subset of transcription factors, which we call cell fate transcription factors, in the regulation of PcG association with target genes. We also speculate about how their deregulation contributes to tumorigenesis.