Targeting the LSD1/KDM1 Family of Lysine Demethylases in Cancer and Other Human Diseases.

Lysine-specific demethylase 1 (LSD1) was the first histone demethylase discovered and the founding member of the flavin-dependent lysine demethylase family (KDM1). The human KDM1 family includes KDM1A and KDM1B, which primarily catalyze demethylation of histone H3K4me1/2. The KDM1 family is involved in epigenetic gene regulation and plays important roles in various biological and disease pathogenesis processes, including cell differentiation, embryonic development, hormone signaling, and carcinogenesis. Malfunction of many epigenetic regulators results in complex human diseases, including cancers. Regulators such as KDM1 have become potential therapeutic targets because of the reversibility of epigenetic control of genome function. Indeed, several classes of KDM1-selective small molecule inhibitors have been developed, some of which are currently in clinical trials to treat various cancers. In this chapter, we review the discovery, biochemical, and molecular mechanisms, atomic structure, genetics, biology, and pathology of the KDM1 family of lysine demethylases. Focusing on cancer, we also provide a comprehensive summary of recently developed KDM1 inhibitors and related preclinical and clinical studies to provide a better understanding of the mechanisms of action and applications of these KDM1-specific inhibitors in therapeutic treatment.

METTL14 regulates chromatin bivalent domains in mouse embryonic stem cells.

METTL14 (methyltransferase-like 14) is an RNA-binding protein that partners with METTL3 to mediate N6-methyladenosine (m6A) methylation. Recent studies identified a function for METTL3 in heterochromatin in mouse embryonic stem cells (mESCs), but the molecular function of METTL14 on chromatin in mESCs remains unclear. Here, we show that METTL14 specifically binds and regulates bivalent domains, which are marked by trimethylation of histone H3 lysine 27 (H3K27me3) and lysine 4 (H3K4me3). Knockout of Mettl14 results in decreased H3K27me3 but increased H3K4me3 levels, leading to increased transcription. We find that bivalent domain regulation by METTL14 is independent of METTL3 or m6A modification. METTL14 enhances H3K27me3 and reduces H3K4me3 by interacting with and probably recruiting the H3K27 methyltransferase polycomb repressive complex 2 (PRC2) and H3K4 demethylase KDM5B to chromatin. Our findings identify an METTL3-independent role of METTL14 in maintaining the integrity of bivalent domains in mESCs, thus indicating a mechanism of bivalent domain regulation in mammals.

Dynamic control of chromatin-associated m6A methylation regulates nascent RNA synthesis.

N6-methyladenosine (m6A) methylation is co-transcriptionally deposited on mRNA, but a possible role of m6A on transcription remains poorly understood. Here, we demonstrate that the METTL3/METTL14/WTAP m6A methyltransferase complex (MTC) is localized to many promoters and enhancers and deposits the m6A modification on nascent transcripts, including pre-mRNAs, promoter upstream transcripts (PROMPTs), and enhancer RNAs. PRO-seq analyses demonstrate that nascent RNAs originating from both promoters and enhancers are significantly decreased in the METTL3-depleted cells. Furthermore, genes targeted by the Integrator complex for premature termination are depleted of METTL3, suggesting a potential antagonistic relationship between METTL3 and Integrator. Consistently, we found the Integrator complex component INTS11 elevated at promoters and enhancers upon loss of MTC or nuclear m6A binders. Taken together, our findings suggest that MTC-mediated m6A modification protects nascent RNAs from Integrator-mediated termination and promotes productive transcription, thus unraveling an unexpected layer of gene regulation imposed by RNA m6A modification.

TET2 Inhibits PD-L1 Gene Expression in Breast Cancer Cells through Histone Deacetylation.

Activation of PD-1/PD-L1 checkpoint is a critical step for the immune evasion of malignant tumors including breast cancer. However, the epigenetic mechanism underlying the aberrant expression of PD-L1 in breast cancer cells remains poorly understood. To investigate the role of TET2 in the regulation of PD-L1 gene expression, quantitative reverse transcription PCR (RT-qPCR), Western blotting, chromatin immunoprecipitation (ChIP) assay and MeDIP/hMeDIP-qPCR were performed on MCF7 and MDA-MB-231 human breast cancer cells. Here, we reported that TET2 depletion upregulated PD-L1 gene expression in MCF7 cells. Conversely, ectopic expression of TET2 inhibited PD-L1 gene expression in MDA-MB-231 cells. Mechanistically, TET2 protein recruits histone deacetylases (HDACs) to PD-L1 gene promoter and orchestrates a repressive chromatin structure to suppress PD-L1 gene transcription, which is likely independent of DNA demethylation. Consistently, treatment with HDAC inhibitors upregulated PD-L1 gene expression in wild-type (WT) but not TET2 KO MCF7 cells. Furthermore, analysis of the CCLE and TCGA data showed a negative correlation between TET2 and PD-L1 expression in breast cancer. Taken together, our results identify a new epigenetic regulatory mechanism of PD-L1 gene transcription, linking the catalytic activity-independent role of TET2 to the anti-tumor immunity in breast cancer.

METTL3 regulates heterochromatin in mouse embryonic stem cells.

METTL3 (methyltransferase-like 3) mediates the N6-methyladenosine (m6A) methylation of mRNA, which affects the stability of mRNA and its translation into protein1. METTL3 also binds chromatin2-4, but the role of METTL3 and m6A methylation in chromatin is not fully understood. Here we show that METTL3 regulates mouse embryonic stem-cell heterochromatin, the integrity of which is critical for silencing retroviral elements and for mammalian development5. METTL3 predominantly localizes to the intracisternal A particle (IAP)-type family of endogenous retroviruses. Knockout of Mettl3 impairs the deposition of multiple heterochromatin marks onto METTL3-targeted IAPs, and upregulates IAP transcription, suggesting that METTL3 is important for the integrity of IAP heterochromatin. We provide further evidence that RNA transcripts derived from METTL3-bound IAPs are associated with chromatin and are m6A-methylated. These m6A-marked transcripts are bound by the m6A reader YTHDC1, which interacts with METTL3 and in turn promotes the association of METTL3 with chromatin. METTL3 also interacts physically with the histone 3 lysine 9 (H3K9) tri-methyltransferase SETDB1 and its cofactor TRIM28, and is important for their localization to IAPs. Our findings demonstrate that METTL3-catalysed m6A modification of RNA is important for the integrity of IAP heterochromatin in mouse embryonic stem cells, revealing a mechanism of heterochromatin regulation in mammals.

The association between monocytic myeloid-derived suppressor cells levels and the anti-tumor efficacy of anti-PD-1 therapy in NSCLC patients.

Monocytic myeloid-derived suppressor cells (M-MDSCs), granulocytic MDSC (G-MDSCs) and regulatory T cells (Tregs) inhibit adaptive anti-tumor immunity and undermine the efficacy of anti-PD-1 therapy. However, the impact of anti-PD-1 treatment on these immunosuppressive cells has not been clearly defined in non-small cell lung cancer (NSCLC). In this retrospective study, 27 advanced NSCLC patients were divided into partial response (PR), stable disease (SD), and progressive disease (PD) groups. The impact of anti-PD-1 therapy on circulating Tregs, G-MDSCs, and M-MDSCs was assessed by flow cytometer. Here, we found that anti-PD-1 treatment boosted circulating Tregs levels, which presented the most remarkable augment during the first two therapeutic cycles, in NSCLC patients. In contrast, anti-PD-1 therapy did not overall change G-MDSCs and M-MDSCs levels. However, the PR group had a higher baseline level of M-MDSCs, which exhibited a significant decrease after the first cycle of anti-PD-1 treatment. Besides, M-MDSCs levels in the PR group were maintained at a low level in the following therapeutic cycles. Consistently, Tregs levels robustly increased in the syngeneic tumor model after anti-mouse PD-1 Ab treatment. Accordingly, M-MDSCs neutralization by anti-mouse ly6c Ab enhanced the anti-tumor efficacy of anti-PD-1 therapy in mice. Finally, the decreased M-MDSCs levels were associated with the enhanced effector CD8+ T cells expansion in the PR group and mice. In conclusion, anti-PD-1 therapy upregulates Tregs levels in NSCLC patients, and the M-MDSC levels are associated with the anti-tumor efficacy of anti-PD-1 treatment. Neutralization of M-MDSCs may be a promising option to augment anti-PD-1 therapy efficacy in NSCLC.

TET2 promotes anti-tumor immunity by governing G-MDSCs and CD8+ T-cell numbers.

The host immune response is a fundamental mechanism for attenuating cancer progression. Here we report a role for the DNA demethylase and tumor suppressor TET2 in host anti-tumor immunity. Deletion of Tet2 in mice elevates IL-6 levels upon tumor challenge. Elevated IL-6 stimulates immunosuppressive granulocytic myeloid-derived suppressor cells (G-MDSCs), which in turn reduce CD8+ T cells upon tumor challenge. Consequently, systematic knockout of Tet2 in mice leads to accelerated syngeneic tumor growth, which is constrained by anti-PD-1 blockade. Removal of G-MDSCs by the anti-mouse Ly6g antibodies restores CD8+ T-cell numbers in Tet2-/- mice and reboots their anti-tumor activity. Importantly, anti-IL-6 antibody treatment blocks the expansion of G-MDSCs and inhibits syngeneic tumor growth. Collectively, these findings reveal a TET2-mediated IL-6/G-MDSCs/CD8+ T-cell immune response cascade that safeguards host adaptive anti-tumor immunity, offering a cell non-autonomous mechanism of TET2 for tumor suppression.

RACK7 recognizes H3.3G34R mutation to suppress expression of MHC class II complex components and their delivery pathway in pediatric glioblastoma.

Histone H3 point mutations have been identified in incurable pediatric brain cancers, but the mechanisms through which these mutations drive tumorigenesis are incompletely understood. Here, we provide evidence that RACK7 (ZMYND8) recognizes the histone H3.3 patient mutation (H3.3G34R) in vitro and in vivo. We show that RACK7 binding to H3.3G34R suppresses transcription of CIITA, which is the master regulator of MHC (major histocompatibility complex) class II molecules and genes involved in vesicular transport of MHC class II molecules to the cell surface, resulting in suppression of MHC class II molecule expression and transport. CRISPR-based knock-in correction of the H3.3G34R mutation in human pediatric glioblastoma (pGBM) cells significantly reduces overall RACK7 chromatin binding and derepresses the same set of genes as does knocking out RACK7 in the H3.3G34R pGBM cells. By demonstrating that H3.3G34R and RACK7 work together, our findings suggest a potential molecular mechanism by which H3.3G34R promotes cancer.

Nono deficiency compromises TET1 chromatin association and impedes neuronal differentiation of mouse embryonic stem cells.

NONO is a DNA/RNA-binding protein, which plays a critical regulatory role during cell stage transitions of mouse embryonic stem cells (mESCs). However, its function in neuronal lineage commitment and the molecular mechanisms of its action in such processes are largely unknown. Here we report that NONO plays a key role during neuronal differentiation of mESCs. Nono deletion impedes neuronal lineage commitment largely due to a failure of up-regulation of specific genes critical for neuronal differentiation. Many of the NONO regulated genes are also DNA demethylase TET1 targeted genes. Importantly, re-introducing wild type NONO to the Nono KO cells, not only restores the normal expression of the majority of NONO/TET1 coregulated genes but also rescues the defective neuronal differentiation of Nono-deficient mESCs. Mechanistically, our data shows that NONO directly interacts with TET1 via its DNA binding domain and recruits TET1 to genomic loci to regulate 5-hydroxymethylcytosine levels. Nono deletion leads to a significant dissociation of TET1 from chromatin and dysregulation of DNA hydroxymethylation of neuronal genes. Taken together, our findings reveal a key role and an epigenetic mechanism of action of NONO in regulation of TET1-targeted neuronal genes, offering new functional and mechanistic understanding of NONO in stem cell functions, lineage commitment and specification.

Kdm1a promotes SCLC progression by transcriptionally silencing the tumor suppressor Rest.

Small cell lung carcinoma (SCLC) is one of the deadliest cancer types, with a 5-year survival rate less than 10%. Kdm1a/Lsd1 has recently been implicated as a potential therapeutic target for SCLC. However, the underlying molecular mechanism by which Kdm1a promotes the oncogenesis of SCLC has not been fully understood. Kdm1a is significantly elevated in most human SCLC specimens, whereas Rest, a tumor suppressor and neuronal repressive transcriptional factor, is typically inactivated. Knock-out of Kdm1a (Kdm1a-KO) in mouse SCLC cell lines resulted in the suppression of cell growth and soft agar colony formation. RNA-Seq analysis of the Kdm1a-KO cells revealed significant repression of a program of neuroendocrine signature genes, and conversely, a significant upregulation of a network of genes capable of inhibiting tumor cell growth. Rest was identified among the top 10 upregulated genes in Kdm1a-KO cells. The treatment of the SCLC cells with Kdm1a demethylase inhibitors resulted in a dramatic up-regulation of Rest similar to the extent of that in Kdm1a-KO cells. Importantly, accompanying the restored expression of the SCLC signature genes, knock-out of Rest in Kdm1a-KO cells rescued the restricted cell growth and soft agar colony formation. Taken together, these novel findings show that Kdm1a is a key transcriptional repressor of Rest, and that suppression of SCLC progression by the targeted inhibition of Kdm1a depends on the reactivation of Rest, suggesting a new strategy for effective SCLC treatment by targeting the Kdm1a/Rest molecular pathway.

Publisher Correction: N6-Methyladenosine methyltransferase ZCCHC4 mediates ribosomal RNA methylation.

In the version of this article originally published, the references were incorrectly re-ordered during production. The hyphen in "N6-methyladenosine" in the title was also superscript. The errors have been corrected in the HTML and PDF versions of the paper.

N6-Methyladenosine methyltransferase ZCCHC4 mediates ribosomal RNA methylation.

N6-Methyladenosine (m6A) RNA modification is present in messenger RNAs (mRNA), ribosomal RNAs (rRNA), and spliceosomal RNAs (snRNA) in humans. Although mRNA m6A modifications have been extensively studied and shown to play critical roles in many cellular processes, the identity of m6A methyltransferases for rRNAs and the function of rRNA m6A modifications are unknown. Here we report a new m6A methyltransferase, ZCCHC4, which primarily methylates human 28S rRNA and also interacts with a subset of mRNAs. ZCCHC4 knockout eliminates m6A4220 modification in 28S rRNA, reduces global translation, and inhibits cell proliferation. We also find that ZCCHC4 protein is overexpressed in hepatocellular carcinoma tumors, and ZCCHC4 knockout significantly reduces tumor size in a xenograft mouse model. Our results highlight the functional significance of an rRNA m6A modification in translation and in tumor biology.

ERα is a negative regulator of PD-L1 gene transcription in breast cancer.

The programmed death-ligand 1 (PD-L1) expression by tumors results in potent antitumor immune suppression through binding to programmed death-1 (PD-1) on T cells and subsequent inhibition of T cells activity. Although recent pathological studies have shown that PD-L1 is actively expressed in certain ERα-negative breast cancer, little is known about whether ER signaling regulates PD-L1 gene expression. Here, we investigated the relationship between ERα and PD-L1 in breast cancer. Analysis of Comprehensive Cell Line Encyclopedia (CCLE) data showed that the average mRNA level of PD-L1 in ERα-positive breast cancer cell lines was significantly lower than that in ERα-negative breast cancer cell lines. E2 treatment inhibited PD-L1 mRNA expression in hormone-depleted ERα-positive MCF7 cells. Moreover, ectopic expression of ERα in triple-negative MDA-MB-231 cells reduced PD-L1 mRNA and protein expression. Consistently, analysis of The Cancer Genome Atlas (TCGA) data revealed an inverse correlation between ERα and PD-L1 expression in ERα-positive breast cancer. Taken together, our results identify ERα as a negative regulator of PD-L1 gene transcription in breast cancer cells, suggesting that ERα loss-of-function may facilitate the immune evasion of breast cancer cells via up-regulation of PD-L1.

Glucose-regulated phosphorylation of TET2 by AMPK reveals a pathway linking diabetes to cancer.

Diabetes is a complex metabolic syndrome that is characterized by prolonged high blood glucose levels and frequently associated with life-threatening complications1,2. Epidemiological studies have suggested that diabetes is also linked to an increased risk of cancer3-5. High glucose levels may be a prevailing factor that contributes to the link between diabetes and cancer, but little is known about the molecular basis of this link and how the high glucose state may drive genetic and/or epigenetic alterations that result in a cancer phenotype. Here we show that hyperglycaemic conditions have an adverse effect on the DNA 5-hydroxymethylome. We identify the tumour suppressor TET2 as a substrate of the AMP-activated kinase (AMPK), which phosphorylates TET2 at serine 99, thereby stabilizing the tumour suppressor. Increased glucose levels impede AMPK-mediated phosphorylation at serine 99, which results in the destabilization of TET2 followed by dysregulation of both 5-hydroxymethylcytosine (5hmC) and the tumour suppressive function of TET2 in vitro and in vivo. Treatment with the anti-diabetic drug metformin protects AMPK-mediated phosphorylation of serine 99, thereby increasing TET2 stability and 5hmC levels. These findings define a novel 'phospho-switch' that regulates TET2 stability and a regulatory pathway that links glucose and AMPK to TET2 and 5hmC, which connects diabetes to cancer. Our data also unravel an epigenetic pathway by which metformin mediates tumour suppression. Thus, this study presents a new model for how a pernicious environment can directly reprogram the epigenome towards an oncogenic state, offering a potential strategy for cancer prevention and treatment.

Zc3h13 Regulates Nuclear RNA m6A Methylation and Mouse Embryonic Stem Cell Self-Renewal.

N6-methyladenosine (m6A) is an abundant modification in eukaryotic mRNA, regulating mRNA dynamics by influencing mRNA stability, splicing, export, and translation. However, the precise m6A regulating machinery still remains incompletely understood. Here we demonstrate that ZC3H13, a zinc-finger protein, plays an important role in modulating RNA m6A methylation in the nucleus. We show that knockdown of Zc3h13 in mouse embryonic stem cell significantly decreases global m6A level on mRNA. Upon Zc3h13 knockdown, a great majority of WTAP, Virilizer, and Hakai translocate to the cytoplasm, suggesting that Zc3h13 is required for nuclear localization of the Zc3h13-WTAP-Virilizer-Hakai complex, which is important for RNA m6A methylation. Finally, Zc3h13 depletion, as does WTAP, Virilizer, or Hakai, impairs self-renewal and triggers mESC differentiation. Taken together, our findings demonstrate that Zc3h13 plays a critical role in anchoring WTAP, Virilizer, and Hakai in the nucleus to facilitate m6A methylation and to regulate mESC self-renewal.

Nono, a Bivalent Domain Factor, Regulates Erk Signaling and Mouse Embryonic Stem Cell Pluripotency.

Nono is a component of the para-speckle, which stores and processes RNA. Mouse embryonic stem cells (mESCs) lack para-speckles, leaving the function of Nono in mESCs unclear. Here, we find that Nono functions as a chromatin regulator cooperating with Erk to regulate mESC pluripotency. We report that Nono loss results in robust self-renewing mESCs with epigenomic and transcriptomic features resembling the 2i (GSK and Erk inhibitors)-induced "ground state." Erk interacts with and is required for Nono localization to a subset of bivalent genes that have high levels of poised RNA polymerase. Nono loss compromises Erk activation and RNA polymerase poising at its target bivalent genes in undifferentiated mESCs, thus disrupting target gene activation and differentiation. These findings argue that Nono collaborates with Erk signaling to regulate the integrity of bivalent domains and mESC pluripotency.

A primary role of TET proteins in establishment and maintenance of De Novo bivalency at CpG islands.

Ten Eleven Translocation (TET) protein-catalyzed 5mC oxidation not only creates novel DNA modifications, such as 5hmC, but also initiates active or passive DNA demethylation. TETs' role in the crosstalk with specific histone modifications, however, is largely elusive. Here, we show that TET2-mediated DNA demethylation plays a primary role in the de novo establishment and maintenance of H3K4me3/H3K27me3 bivalent domains underlying methylated DNA CpG islands (CGIs). Overexpression of wild type (WT), but not catalytic inactive mutant (Mut), TET2 in low-TET-expressing cells results in an increase in the level of 5hmC with accompanying DNA demethylation at a subset of CGIs. Most importantly, this alteration is sufficient in making de novo bivalent domains at these loci. Genome-wide analysis reveals that these de novo synthesized bivalent domains are largely associated with a subset of essential developmental gene promoters, which are located within CGIs and are previously silenced due to DNA methylation. On the other hand, deletion of Tet1 and Tet2 in mouse embryonic stem (ES) cells results in an apparent loss of H3K27me3 at bivalent domains, which are associated with a particular set of key developmental gene promoters. Collectively, this study demonstrates the critical role of TET proteins in regulating the crosstalk between two key epigenetic mechanisms, DNA methylation and histone methylation (H3K4me3 and H3K27me3), particularly at CGIs associated with developmental genes.