TAK1 inhibition leads to RIPK1-dependent apoptosis in immune-activated cancers.

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 TGFß 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.

Vitamin C activates young LINE-1 elements in mouse embryonic stem cells via H3K9me3 demethylation.

BACKGROUND: Vitamin C (vitC) enhances the activity of 2-oxoglutarate-dependent dioxygenases, including TET enzymes, which catalyse DNA demethylation, and Jumonji-domain histone demethylases. The epigenetic remodelling promoted by vitC improves the efficiency of induced pluripotent stem cell derivation, and is required to attain a ground-state of pluripotency in embryonic stem cells (ESCs) that closely mimics the inner cell mass of the early blastocyst. However, genome-wide DNA and histone demethylation can lead to upregulation of transposable elements (TEs), and it is not known how vitC addition in culture media affects TE expression in pluripotent stem cells. RESULTS: Here we show that vitC increases the expression of several TE families, including evolutionarily young LINE-1 (L1) elements, in mouse ESCs. We find that TET activity is dispensable for L1 upregulation, and that instead it occurs largely as a result of H3K9me3 loss mediated by KDM4A/C histone demethylases. Despite increased L1 levels, we did not detect increased somatic insertion rates in vitC-treated cells. Notably, treatment of human ESCs with vitC also increases L1 protein levels, albeit through a distinct, post-transcriptional mechanism. CONCLUSION: VitC directly modulates the expression of mouse L1s and other TEs through epigenetic mechanisms, with potential for downstream effects related to the multiple emerging roles of L1s in cellular function.

Targeting RIOK2 ATPase activity leads to decreased protein synthesis and cell death in acute myeloid leukemia.

Novel therapies for the treatment of acute myeloid leukemia (AML) are urgently needed, because current treatments do not cure most patients with AML. We report a domain-focused, kinome-wide CRISPR-Cas9 screening that identified protein kinase targets for the treatment of AML, which led to the identification of Rio-kinase 2 (RIOK2) as a potential novel target. Loss of RIOK2 led to a decrease in protein synthesis and to ribosomal instability followed by apoptosis in leukemic cells, but not in fibroblasts. Moreover, the ATPase function of RIOK2 was necessary for cell survival. When a small-molecule inhibitor was used, pharmacological inhibition of RIOK2 similarly led to loss of protein synthesis and apoptosis and affected leukemic cell growth in vivo. Our results provide proof of concept for targeting RIOK2 as a potential treatment of patients with AML.

The KDM4/JMJD2 histone demethylases are required for hematopoietic stem cell maintenance.

KDM4/JMJD2 are H3K9- and H3K36-specific demethylases, which are considered promising therapeutic targets for the treatment of acute myeloid leukemia (AML) harboring MLL translocations. Here, we investigate the long-term effects of depleting KDM4 activity on normal hematopoiesis to probe potential side effects of continuous inhibition of these enzymes. Utilizing conditional Kdm4a/Kdm4b/Kdm4c triple-knockout mice, we show that KDM4 activity is required for hematopoietic stem cell (HSC) maintenance in vivo. The knockout of the KDM4 demethylases leads to accumulation of H3K9me3 on transcription start sites and the corresponding downregulation of expression of several genes in HSCs. We show that 2 of these genes, Taf1b and Nom1, are essential for the maintenance of hematopoietic cells. Taken together, our results show that the KDM4 demethylases are required for the expression of genes essential for the long-term maintenance of normal hematopoiesis.

The Lysine Demethylase KDM5B Regulates Islet Function and Glucose Homeostasis.

AIMS: Posttranslational modifications of histones and transcription factors regulate gene expression and are implicated in beta-cell failure and diabetes. We have recently shown that preserving H3K27 and H3K4 methylation using the lysine demethylase inhibitor GSK-J4 reduces cytokine-induced destruction of beta-cells and improves beta-cell function. Here, we investigate the therapeutic potential of GSK-J4 to prevent diabetes development and examine the importance of H3K4 methylation for islet function. MATERIALS AND METHODS: We used two mouse models of diabetes to investigate the therapeutic potential of GSK-J4. To clarify the importance of H3K4 methylation, we characterized a mouse strain with knockout (KO) of the H3K4 demethylase KDM5B. RESULTS: GSK-J4 administration failed to prevent the development of experimental diabetes induced by multiple low-dose streptozotocin or adoptive transfer of splenocytes from acutely diabetic NOD to NODscid mice. KDM5B-KO mice were growth retarded with altered body composition, had low IGF-1 levels, and exhibited reduced insulin secretion. Interestingly, despite secreting less insulin, KDM5B-KO mice were able to maintain normoglycemia following oral glucose tolerance test, likely via improved insulin sensitivity, as suggested by insulin tolerance testing and phosphorylation of proteins belonging to the insulin signaling pathway. When challenged with high-fat diet, KDM5B-deficient mice displayed similar weight gain and insulin sensitivity as wild-type mice. CONCLUSION: Our results show a novel role of KDM5B in metabolism, as KDM5B-KO mice display growth retardation and improved insulin sensitivity.

Jarid2 binds mono-ubiquitylated H2A lysine 119 to mediate crosstalk between Polycomb complexes PRC1 and PRC2.

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.

Jmjd2/Kdm4 demethylases are required for expression of Il3ra and survival of acute myeloid leukemia cells.

Acute myeloid leukemias (AMLs) with a rearrangement of the mixed-linage leukemia (MLL) gene are aggressive hematopoietic malignancies. Here, we explored the feasibility of using the H3K9- and H3K36-specific demethylases Jmjd2/Kdm4 as putative drug targets in MLL-AF9 translocated leukemia. Using Jmjd2a, Jmjd2b, and Jmjd2c conditional triple-knockout mice, we show that Jmjd2/Kdm4 activities are required for MLL-AF9 translocated AML in vivo and in vitro. We demonstrate that expression of the interleukin 3 receptor α (Il3ra also known as Cd123) subunit is dependent on Jmjd2/Kdm4 through a mechanism involving removal of H3K9me3 from the promoter of the Il3ra gene. Importantly, ectopic expression of Il3ra in Jmjd2/Kdm4 knockout cells alleviates the requirement of Jmjd2/Kdm4 for the survival of AML cells, showing that Il3ra is a critical downstream target of Jmjd2/Kdm4 in leukemia. These results suggest that the JMJD2/KDM4 proteins are promising drug targets for the treatment of AML.

Continual removal of H3K9 promoter methylation by Jmjd2 demethylases is vital for ESC self-renewal and early development.

Chromatin-associated proteins are essential for the specification and maintenance of cell identity. They exert these functions through modulating and maintaining transcriptional patterns. To elucidate the functions of the Jmjd2 family of H3K9/H3K36 histone demethylases, we generated conditional Jmjd2a/Kdm4a, Jmjd2b/Kdm4b and Jmjd2c/Kdm4c/Gasc1 single, double and triple knockout mouse embryonic stem cells (ESCs). We report that while individual Jmjd2 family members are dispensable for ESC maintenance and embryogenesis, combined deficiency for specifically Jmjd2a and Jmjd2c leads to early embryonic lethality and impaired ESC self-renewal, with spontaneous differentiation towards primitive endoderm under permissive culture conditions. We further show that Jmjd2a and Jmjd2c both localize to H3K4me3-positive promoters, where they have widespread and redundant roles in preventing accumulation of H3K9me3 and H3K36me3. Jmjd2 catalytic activity is required for ESC maintenance, and increased H3K9me3 levels in knockout ESCs compromise the expression of several Jmjd2a/c targets, including genes that are important for ESC self-renewal. Thus, continual removal of H3K9 promoter methylation by Jmjd2 demethylases represents a novel mechanism ensuring transcriptional competence and stability of the pluripotent cell identity.

The demethylase JMJD2C localizes to H3K4me3-positive transcription start sites and is dispensable for embryonic development.

The histone demethylase JMJD2C, also known as KDM4C/GASC1, has activity against methylated H3K9 and H3K36 and is amplified and/or overexpressed in human cancers. By the generation of Jmjd2c knockout mice, we demonstrate that loss of Jmjd2c is compatible with cellular proliferation, embryonic stem cell (ESC) self-renewal, and embryonic development. Moreover, we report that JMJD2C localizes to H3K4me3-positive transcription start sites in both primary cells and in the human carcinoma KYSE150 cell line containing an amplification of the JMJD2C locus. Binding is dependent on the double Tudor domain of JMJD2C, which recognizes H3K4me3 but not H4K20me2/me3 in vitro, showing a binding specificity different from that of the double Tudor domains of JMJD2A and JMJD2B. Depletion of JMJD2C in KYSE150 cells has a modest effect on H3K9me3 and H3K36me3 levels but impairs proliferation and leads to deregulated expression of a subset of target genes involved in cell cycle progression. Taking these findings together, we show that JMJD2C is targeted to H3K4me3-positive transcription start sites, where it can contribute to transcriptional regulation, and report that the putative oncogene JMJD2C generally is not required for cellular proliferation or embryonic development.

Histone lysine demethylases as targets for anticancer therapy.

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.

Inhibitors of histone demethylases.

Methylated lysines are important epigenetic marks. The enzymes involved in demethylation have recently been discovered and found to be involved in cancer development and progression. Despite the relative recent discovery of these enzymes a number of inhibitors have already appeared. Most of the inhibitors are either previously reported inhibitors of related enzymes or compounds derived from these. Development in terms of selectivity and potency is still pertinent. Several reports on the development of functional assays have been published.

The H3K27me3 demethylase JMJD3 contributes to the activation of the INK4A-ARF locus in response to oncogene- and stress-induced senescence.

The tumor suppressor proteins p16INK4A and p14ARF, encoded by the INK4A-ARF locus, are key regulators of cellular senescence. The locus is epigenetically silenced by the repressive H3K27me3 mark in normally growing cells, but becomes activated in response to oncogenic stress. Here, we show that expression of the histone H3 Lys 27 (H3K27) demethylase JMJD3 is induced upon activation of the RAS-RAF signaling pathway. JMJD3 is recruited to the INK4A-ARF locus and contributes to the transcriptional activation of p16INK4A in human diploid fibroblasts. Additionally, inhibition of Jmjd3 expression in mouse embryonic fibroblasts results in suppression of p16Ink4a and p19Arf expression and in their immortalization.

Coordinated regulation of transcriptional repression by the RBP2 H3K4 demethylase and Polycomb-Repressive Complex 2.

Polycomb group (PcG) proteins regulate important cellular processes such as embryogenesis, cell proliferation, and stem cell self-renewal through the transcriptional repression of genes determining cell fate decisions. The Polycomb-Repressive Complex 2 (PRC2) is highly conserved during evolution, and its intrinsic histone H3 Lys 27 (K27) trimethylation (me3) activity is essential for PcG-mediated transcriptional repression. Here, we show a functional interplay between the PRC2 complex and the H3K4me3 demethylase Rbp2 (Jarid1a) in mouse embryonic stem (ES) cells. By genome-wide location analysis we found that Rbp2 is associated with a large number of PcG target genes in mouse ES cells. We show that the PRC2 complex recruits Rbp2 to its target genes, and that this interaction is required for PRC2-mediated repressive activity during ES cell differentiation. Taken together, these results demonstrate an elegant mechanism for repression of developmental genes by the coordinated regulation of epigenetic marks involved in repression and activation of transcription.

Erasing the methyl mark: histone demethylases at the center of cellular differentiation and disease.

The enzymes catalyzing lysine and arginine methylation of histones are essential for maintaining transcriptional programs and determining cell fate and identity. Until recently, histone methylation was regarded irreversible. However, within the last few years, several families of histone demethylases erasing methyl marks associated with gene repression or activation have been identified, underscoring the plasticity and dynamic nature of histone methylation. Recent discoveries have revealed that histone demethylases take part in large multiprotein complexes synergizing with histone deacetylases, histone methyltransferases, and nuclear receptors to control developmental and transcriptional programs. Here we review the emerging biochemical and biological functions of the histone demethylases and discuss their potential involvement in human diseases, including cancer.

The emerging functions of histone demethylases.

Epigenetic information refers to heritable changes in gene function that are stable between cell divisions but which is not a result of changes in the DNA sequence. Part of the epigenetic mechanism has been ascribed to modifications of histones or DNA that affects the transcription of specific genes. In this context, post-translational modifications of histone tails, in particular methylation of lysines, are regarded as important for the storage of epigenetic information. Regulation of this information plays an important role during cellular differentiation where cells with different characteristic features evolve from the same ancestor, despite identical genomic material. The characterization of several enzymes catalyzing histone lysine methylation have supported this concept by showing the requirement of these enzymes for normal development and their involvement in diseases such as cancer. The recent identification of proteins with histone demethylase activity has shown that the methylated mark is much more dynamic than previously anticipated, thereby potentially challenging the concept of histone-methylation in stable epigenetic programming.

UTX and JMJD3 are histone H3K27 demethylases involved in HOX gene regulation and development.

The trithorax and the polycomb group proteins are chromatin modifiers, which play a key role in the epigenetic regulation of development, differentiation and maintenance of cell fates. The polycomb repressive complex 2 (PRC2) mediates transcriptional repression by catalysing the di- and tri-methylation of Lys 27 on histone H3 (H3K27me2/me3). Owing to the essential role of the PRC2 complex in repressing a large number of genes involved in somatic processes, the H3K27me3 mark is associated with the unique epigenetic state of stem cells. The rapid decrease of the H3K27me3 mark during specific stages of embryogenesis and stem-cell differentiation indicates that histone demethylases specific for H3K27me3 may exist. Here we show that the human JmjC-domain-containing proteins UTX and JMJD3 demethylate tri-methylated Lys 27 on histone H3. Furthermore, we demonstrate that ectopic expression of JMJD3 leads to a strong decrease of H3K27me3 levels and causes delocalization of polycomb proteins in vivo. Consistent with the strong decrease in H3K27me3 levels associated with HOX genes during differentiation, we show that UTX directly binds to the HOXB1 locus and is required for its activation. Finally mutation of F18E9.5, a Caenorhabditis elegans JMJD3 orthologue, or inhibition of its expression, results in abnormal gonad development. Taken together, these results suggest that H3K27me3 demethylation regulated by UTX/JMJD3 proteins is essential for proper development. Moreover, the recent demonstration that UTX associates with the H3K4me3 histone methyltransferase MLL2 (ref. 8) supports a model in which the coordinated removal of repressive marks, polycomb group displacement, and deposition of activating marks are important for the stringent regulation of transcription during cellular differentiation.

RBP2 belongs to a family of demethylases, specific for tri-and dimethylated lysine 4 on histone 3.

Methylation of histones has been regarded as a stable modification defining the epigenetic program of the cell, which regulates chromatin structure and transcription. However, the recent discovery of histone demethylases has challenged the stable nature of histone methylation. Here we demonstrate that the JARID1 proteins RBP2, PLU1, and SMCX are histone demethylases specific for di- and trimethylated histone 3 lysine 4 (H3K4). Consistent with a role for the JARID1 Drosophila homolog Lid in regulating expression of homeotic genes during development, we show that RBP2 is displaced from Hox genes during embryonic stem (ES) cell differentiation correlating with an increase of their H3K4me3 levels and expression. Furthermore, we show that mutation or RNAi depletion of the C. elegans JARID1 homolog rbr-2 leads to increased levels of H3K4me3 during larval development and defects in vulva formation. Taken together, these results suggest that H3K4me3/me2 demethylation regulated by the JARID1 family plays an important role during development.

The putative oncogene GASC1 demethylates tri- and dimethylated lysine 9 on histone H3.

Methylation of lysine and arginine residues on histone tails affects chromatin structure and gene transcription. Tri- and dimethylation of lysine 9 on histone H3 (H3K9me3/me2) is required for the binding of the repressive protein HP1 and is associated with heterochromatin formation and transcriptional repression in a variety of species. H3K9me3 has long been regarded as a 'permanent' epigenetic mark. In a search for proteins and complexes interacting with H3K9me3, we identified the protein GASC1 (gene amplified in squamous cell carcinoma 1), which belongs to the JMJD2 (jumonji domain containing 2) subfamily of the jumonji family, and is also known as JMJD2C. Here we show that three members of this subfamily of proteins demethylate H3K9me3/me2 in vitro through a hydroxylation reaction requiring iron and alpha-ketoglutarate as cofactors. Furthermore, we demonstrate that ectopic expression of GASC1 or other JMJD2 members markedly decreases H3K9me3/me2 levels, increases H3K9me1 levels, delocalizes HP1 and reduces heterochromatin in vivo. Previously, GASC1 was found to be amplified in several cell lines derived from oesophageal squamous carcinomas, and in agreement with a contribution of GASC1 to tumour development, inhibition of GASC1 expression decreases cell proliferation. Thus, in addition to identifying GASC1 as a histone trimethyl demethylase, we suggest a model for how this enzyme might be involved in cancer development, and propose it as a target for anti-cancer therapy.

A novel repressive E2F6 complex containing the polycomb group protein, EPC1, that interacts with EZH2 in a proliferation-specific manner.

The transcriptional repressor E2F6 has been identified as a component of two distinct polycomb group protein (PcG)-containing complexes, suggesting a mechanism for the recruitment of repressive complexes to target sequences in DNA. Whereas one complex is involved in the repression of classic E2F target genes in G0, a role for E2F6 within the cell cycle has yet to be defined. We searched for novel E2F6-binding proteins using a yeast two-hybrid screen and identified the PcG protein, EPC1. We showed that, both in vitro and in vivo, E2F6, DP1, and EPC1 form a stable core complex with repressive activity. Furthermore, we identified the proliferation-specific PcG, EZH2, as an EPC1-interacting protein. Using affinity purification, we showed that E2F6, DP1, EPC1, EZH2, and Sin3B co-elute, suggesting the identification of a novel E2F6 complex that exists in vivo in both normal and transformed human cell lines. EZH2 is required for cellular proliferation and consistent with this, EZH2 elutes with the E2F6-EPC1 complex only in proliferating cells. Thus we have identified a novel E2F6-PcG complex (E2F6-EPC1) that interacts with EZH2 and may regulate genes required for cell cycle progression.

Conditional E2F1 activation in transgenic mice causes testicular atrophy and dysplasia mimicking human CIS.

E2F1 is a crucial downstream effector of the retinoblastoma protein (pRB) pathway. To address the consequences of short-term increase in E2F1 activity in adult tissues, we generated transgenic mice expressing the human E2F1 protein fused to the oestrogen receptor (ER) ligand-binding domain. The expression of the ER-E2F1 fusion protein, which is inactive in the absence of 4-hydroxy tamoxifen (OHT), was targeted to the testes. We show that short-term activation of E2F1 results in activation of E2F target genes and apoptosis of germ cells. Consistent with our previously published results, the apoptotic response was independent of p53. Persistent E2F1 activation for 3 weeks led to massive apoptosis and severe testicular atrophy with seminiferous tubules containing only Sertoli cells and clusters of undifferentiated spermatogonia. The latter showed high expression of ER-E2F1 and excessive mitotic activity, including atypical mitoses. In addition, gonocyte-like dysplastic germ cells, resembling carcinoma in situ (CIS) cells in humans, appeared. Our results show that a relatively short period of deregulated E2F1 activity in testicles can induce premalignant changes. Moreover, we demonstrate the feasibility of tissue-specific expression of conditional ER-E2F1 in transgenic mice.