Methylation of histone H3 lysine 36 is a barrier for therapeutic interventions of head and neck squamous cell carcinoma.

Approximately 20% of head and neck squamous cell carcinomas (HNSCCs) exhibit reduced methylation on lysine 36 of histone H3 (H3K36me) due to mutations in histone methylase NSD1 or a lysine-to-methionine mutation in histone H3 (H3K36M). Whether such alterations of H3K36me can be exploited for therapeutic interventions is still unknown. Here, we show that HNSCC models expressing H3K36M can be divided into two groups: those that display aberrant accumulation of H3K27me3 and those that maintain steady levels of H3K27me3. The former group exhibits reduced proliferation, genome instability, and heightened sensitivity to genotoxic agents like PARP1/2 inhibitors. Conversely, H3K36M HNSCC models with constant H3K27me3 levels lack these characteristics unless H3K27me3 is elevated by DNA hypomethylating agents or inhibiting H3K27me3 demethylases KDM6A/B. Mechanistically, H3K36M reduces H3K36me by directly impeding the activities of the histone methyltransferase NSD3 and the histone demethylase LSD2. Notably, aberrant H3K27me3 levels induced by H3K36M expression are not a bona fide epigenetic mark because they require continuous expression of H3K36M to be inherited. Moreover, increased sensitivity to PARP1/2 inhibitors in H3K36M HNSCC models depends solely on elevated H3K27me3 levels and diminishing BRCA1- and FANCD2-dependent DNA repair. Finally, a PARP1/2 inhibitor alone reduces tumor burden in a H3K36M HNSCC xenograft model with elevated H3K27me3, whereas in a model with consistent H3K27me3, a combination of PARP1/2 inhibitors and agents that up-regulate H3K27me3 proves to be successful. These findings underscore the crucial balance between H3K36 and H3K27 methylation in maintaining genome instability, offering new therapeutic options for patients with H3K36me-deficient tumors.

Methylation of histone H3 lysine 36 is a barrier for therapeutic interventions of head and neck squamous cell carcinoma.

Approximately 20% of head and neck squamous cell carcinomas (HNSCC) exhibit reduced methylation on lysine 36 of histone H3 (H3K36me) due to mutations in histone methylase NSD1 or a lysine-to-methionine mutation in histone H3 (H3K36M). Whether such alterations of H3K36me can be exploited for therapeutic interventions is still unknown. Here, we show that HNSCC models expressing H3K36M can be divided into two groups: those that display aberrant accumulation of H3K27me3 and those that maintain steady levels of H3K27me3. The first group shows decreased proliferation, genome instability, and increased sensitivity to genotoxic agents, such as PARP1/2 inhibitors. In contrast, the H3K36M HNSCC models with steady H3K27me3 levels do not exhibit these characteristics unless H3K27me3 levels are elevated, either by DNA hypomethylating agents or by inhibiting the H3K27me3 demethylases KDM6A/B. Mechanistically, we found that H3K36M reduces H3K36me by directly impeding the activities of the histone methyltransferase NSD3 and the histone demethylase LSD2. Notably, we found that aberrant H3K27me3 levels induced by H3K36M expression is not a bona fide epigenetic mark in HNSCC since it requires continuous expression of H3K36M to be inherited. Moreover, increased sensitivity of H3K36M HNSCC models to PARP1/2 inhibitors solely depends on the increased H3K27me3 levels. Indeed, aberrantly high H3K27me3 levels decrease BRCA1 and FANCD2-dependent DNA repair, resulting in higher sensitivity to DNA breaks and replication stress. Finally, in support of our in vitro findings, a PARP1/2 inhibitor alone reduce tumor burden in a H3K36M HNSCC xenograft model with elevated H3K27me3, whereas in a H3K36M HNSCC xenograft model with consistent H3K27me3 levels, a combination of PARP1/2 inhibitors and agents that upregulate H3K27me3 proves to be successful. In conclusion, our findings underscore a delicate balance between H3K36 and H3K27 methylation, essential for maintaining genome stability. This equilibrium presents promising therapeutic opportunities for patients with H3K36me-deficient tumors.

The SWI/SNF chromatin-remodeling subunit DPF2 facilitates NRF2-dependent antiinflammatory and antioxidant gene expression.

During emergency hematopoiesis, hematopoietic stem cells (HSCs) rapidly proliferate to produce myeloid and lymphoid effector cells, a response that is critical against infection or tissue injury. If unresolved, this process leads to sustained inflammation, which can cause life-threatening diseases and cancer. Here, we identify a role of double PHD fingers 2 (DPF2) in modulating inflammation. DPF2 is a defining subunit of the hematopoiesis-specific BAF (SWI/SNF) chromatin-remodeling complex, and it is mutated in multiple cancers and neurological disorders. We uncovered that hematopoiesis-specific Dpf2-KO mice developed leukopenia, severe anemia, and lethal systemic inflammation characterized by histiocytic and fibrotic tissue infiltration resembling a clinical hyperinflammatory state. Dpf2 loss impaired the polarization of macrophages responsible for tissue repair, induced the unrestrained activation of Th cells, and generated an emergency-like state of HSC hyperproliferation and myeloid cell-biased differentiation. Mechanistically, Dpf2 deficiency resulted in the loss of the BAF catalytic subunit BRG1 from nuclear factor erythroid 2-like 2-controlled (NRF2-controlled) enhancers, impairing the antioxidant and antiinflammatory transcriptional response needed to modulate inflammation. Finally, pharmacological reactivation of NRF2 suppressed the inflammation-mediated phenotypes and lethality of Dpf2Δ/Δ mice. Our work establishes an essential role of the DPF2-BAF complex in licensing NRF2-dependent gene expression in HSCs and immune effector cells to prevent chronic inflammation.

Endocrine resistance and breast cancer plasticity are controlled by CoREST.

Resistance to cancer treatment remains a major clinical hurdle. Here, we demonstrate that the CoREST complex is a key determinant of endocrine resistance and ER+ breast cancer plasticity. In endocrine-sensitive cells, CoREST is recruited to regulatory regions co-bound to ERα and FOXA1 to regulate the estrogen pathway. In contrast, during temporal reprogramming towards a resistant state, CoREST is recruited to AP-1 sites. In reprogrammed cells, CoREST favors chromatin opening, cJUN binding to chromatin, and gene activation by controlling SWI/SNF recruitment independently of the demethylase activity of the CoREST subunit LSD1. Genetic and pharmacological CoREST inhibition reduces tumorigenesis and metastasis of endocrine-sensitive and endocrine-resistant xenograft models. Consistently, CoREST controls a gene signature involved in invasiveness in clinical breast tumors resistant to endocrine therapies. Our studies reveal CoREST functions that are co-opted to drive cellular plasticity and resistance to endocrine therapies and tumorigenesis, thus establishing CoREST as a potential therapeutic target for the treatment of advanced breast cancer.

DDX41 expression is associated with tumor necrosis in clear cell renal cell carcinoma and in cooperation with VHL loss leads to worse prognosis.

BACKGROUND: Histologic tumor necrosis (TN) is a well-established independent prognostic indicator in patients treated surgically for clear cell renal cell carcinoma (ccRCC). However, the precise mechanisms by which TN alters disease progression remain unknown. The DEAD-box protein DDX41, a member of a large family of helicases, has been characterized as a pattern recognition receptor against an array of double-stranded (ds)DNA produced from bacteria, dsDNA viruses, and nearby cells that have released dsDNA fragments through necrosis. We hypothesized that DDX41 expression may be upregulated in ccRCC with TN, leading to worse prognosis. METHODS: Relationship between the presence of TN and DDX41 expression were examined using The Cancer Genome Atlas data sets or using ccRCC samples in our institution. Further, the molecular functions of DDX41 were investigated with human ccRCC cells. RESULTS: The presence of TN was significantly associated with the upregulation of mRNA and protein expression of DDX41 in the 2different patient cohorts with ccRCC. In addition, the mRNA and protein expression levels of DDX41 revealed a worse prognosis. In vitro analyses with ccRCC cells revealed that DDX41 expression promotes tumor-promoting activity. Furthermore, VHL loss, 1of the most common features in ccRCC, was shown to play an extremely important role in increasing the expression of the CXCL family in DDX41-expressing ccRCC, leading to the acquisition of a worse malignant phenotype. CONCLUSIONS: DDX41 expression is associated with TN in ccRCC and leads to a worse prognosis in cooperation with VHL loss.

Expression and Function of Nicotinic Acetylcholine Receptors in Induced Regulatory T Cells.

A contribution of the cholinergic system to immune cell function has been suggested, though the role of nicotine and its receptors in T cells, especially regulatory T (Treg) cells, is unclear. We herein investigated the expression and function of nicotinic acetylcholine receptors (nAChRs) in murine-induced Treg (iTreg) cells. Upon differentiation of naive BALB/c T cells into iTreg cells and other T-cell subsets, the effect of nicotine on cytokine production and proliferation of iTreg cells was examined. The expression of nAChRs and its regulatory mechanisms were comparatively analyzed among T-cell subsets. Stimulation-induced transforming growth factor-ß1 (TGF-ß1) production of iTreg cells was suppressed by nicotine, whereas interleukin (IL)-10 production and proliferation was not affected. α2-, α5-, α9-, and ß2-nAChRs were differentially expressed in naive, Th1, Th2, Th9, Th17, and iTreg cells. Among these cell types, the α9-nAChR was particularly upregulated in iTreg cells via its gene promoter, but not through tri-methylation at the 4th lysine residue of the histone H3-dependent mechanisms. We conclude that the immunoregulatory role of Treg cells is modified by the cholinergic system, probably through the characteristic expression of nAChRs.

Epigenetic mechanisms in breast cancer therapy and resistance.

The majority of breast cancers express the estrogen receptor (ERα) and agents targeting this pathway represent the main treatment modality. Endocrine therapy has proven successful in the treatment of hormone-responsive breast cancer since its early adoption in the 1940s as an ablative therapy. Unfortunately, therapeutic resistance arises, leading to disease recurrence and relapse. Recent studies increased our understanding in how changes to the chromatin landscape and deregulation of epigenetic factors orchestrate the resistant phenotype. Here, we will discuss how the epigenome is an integral determinant in hormone therapy response and why epigenetic factors are promising targets for overcoming clinical resistance.

UTX maintains the functional integrity of the murine hematopoietic system by globally regulating aging-associated genes.

Epigenetic regulation is essential for the maintenance of the hematopoietic system, and its deregulation is implicated in hematopoietic disorders. In this study, UTX, a demethylase for lysine 27 on histone H3 (H3K27) and a component of COMPASS-like and SWI/SNF complexes, played an essential role in the hematopoietic system by globally regulating aging-associated genes. Utx-deficient (UtxΔ/Δ) mice exhibited myeloid skewing with dysplasia, extramedullary hematopoiesis, impaired hematopoietic reconstituting ability, and increased susceptibility to leukemia, which are the hallmarks of hematopoietic aging. RNA-sequencing (RNA-seq) analysis revealed that Utx deficiency converted the gene expression profiles of young hematopoietic stem-progenitor cells (HSPCs) to those of aged HSPCs. Utx expression in hematopoietic stem cells declined with age, and UtxΔ/Δ HSPCs exhibited increased expression of an aging-associated marker, accumulation of reactive oxygen species, and impaired repair of DNA double-strand breaks. Pathway and chromatin immunoprecipitation analyses coupled with RNA-seq data indicated that UTX contributed to hematopoietic homeostasis mainly by maintaining the expression of genes downregulated with aging via demethylase-dependent and -independent epigenetic programming. Of note, comparison of pathway changes in UtxΔ/Δ HSPCs, aged muscle stem cells, aged fibroblasts, and aged induced neurons showed substantial overlap, strongly suggesting common aging mechanisms among different tissue stem cells.

Kdm6a Deficiency Activates Inflammatory Pathways, Promotes M2 Macrophage Polarization, and Causes Bladder Cancer in Cooperation with p53 Dysfunction.

PURPOSE: Epigenetic deregulation is deeply implicated in the pathogenesis of bladder cancer. KDM6A (Lysine (K)-specific demethylase 6A) is a histone modifier frequently mutated in bladder cancer. However, the molecular mechanisms of how KDM6A deficiency contributes to bladder cancer development remains largely unknown. We hypothesized that clarification of the pathogenic mechanisms underlying KDM6A-mutated bladder cancer can help in designing new anticancer therapies. EXPERIMENTAL DESIGN: We generated mice lacking Kdm6a in the urothelium and crossed them with mice heterozygous for p53, whose mutation/deletion significantly overlaps with the KDM6A mutation in muscle-invasive bladder cancer (MIBC). In addition, BBN (N-butyl-N-(4-hydroxybutyl) nitrosamine), a cigarette smoke-like mutagen, was used as a tumor-promoting agent. Isolated urothelia were subjected to phenotypic, pathologic, molecular, and cellular analyses. The clinical relevance of our findings was further analyzed using genomic and clinical data of patients with MIBC. RESULTS: We found that Kdm6a deficiency activated cytokine and chemokine pathways, promoted M2 macrophage polarization, increased cancer stem cells and caused bladder cancer in cooperation with p53 haploinsufficiency. We also found that BBN treatment significantly enhanced the expression of proinflammatory molecules and accelerated disease development. Human bladder cancer samples with decreased KDM6A expression also showed activated proinflammatory pathways. Notably, dual inhibition of IL6 and chemokine (C-C motif) ligand 2, upregulated in response to Kdm6a deficiency, efficiently suppressed Kdm6a-deficient bladder cancer cell growth. CONCLUSIONS: Our findings provide insights into multistep carcinogenic processes of bladder cancer and suggest molecular targeted therapeutic approaches for patients with bladder cancer with KDM6A dysfunction.

Acquired expression of CblQ367P in mice induces dysplastic myelopoiesis mimicking chronic myelomonocytic leukemia.

Chronic myelomonocytic leukemia (CMML) is a hematological malignancy characterized by uncontrolled proliferation of dysplastic myelomonocytes and frequent progression to acute myeloid leukemia (AML). We identified mutations in the Cbl gene, which encodes a negative regulator of cytokine signaling, in a subset of CMML patients. To investigate the contribution of mutant Cbl in CMML pathogenesis, we generated conditional knockin mice for Cbl that express wild-type Cbl in a steady state and inducibly express CblQ367P , a CMML-associated Cbl mutant. CblQ367P mice exhibited sustained proliferation of myelomonocytes, multilineage dysplasia, and splenomegaly, which are the hallmarks of CMML. The phosphatidylinositol 3-kinase (PI3K)-AKT and JAK-STAT pathways were constitutively activated in CblQ367P hematopoietic stem cells, which promoted cell cycle progression and enhanced chemokine-chemokine receptor activity. Gem, a gene encoding a GTPase that is upregulated by CblQ367P , enhanced hematopoietic stem cell activity and induced myeloid cell proliferation. In addition, Evi1, a gene encoding a transcription factor, was found to cooperate with CblQ367P and progress CMML to AML. Furthermore, targeted inhibition for the PI3K-AKT and JAK-STAT pathways efficiently suppressed the proliferative activity of CblQ367P -bearing CMML cells. Our findings provide insights into the molecular mechanisms underlying mutant Cbl-induced CMML and propose a possible molecular targeting therapy for mutant Cbl-carrying CMML patients.

Propagation of trimethylated H3K27 regulated by polycomb protein EED is required for embryogenesis, hematopoietic maintenance, and tumor suppression.

Polycomb repressive complex 2 (PRC2) catalyzes the monomethylation, dimethylation, and trimethylation of histone H3 Lys27 (H3K27) and acts as a central epigenetic regulator that marks the repressive chromatin domain. Embryonic ectoderm development (EED), an essential component of PRC2, interacts with trimethylated H3K27 (H3K27me3) through the aromatic cage structure composed of its three aromatic amino acids, Phe97, Trp364, and Tyr365. This interaction allosterically activates the histone methyltransferase activity of PRC2 and thereby propagates repressive histone marks. In this study, we report the analysis of knock-in mice harboring the myeloid disorder-associated EED Ile363Met (I363M) mutation, analogous to the EED aromatic cage mutants. The I363M homozygotes displayed a remarkable and preferential reduction of H3K27me3 and died at midgestation. The heterozygotes increased the clonogenic capacity and bone marrow repopulating activity of hematopoietic stem/progenitor cells (HSPCs) and were susceptible to leukemia. Lgals3, a PRC2 target gene encoding a multifunctional galactose-binding lectin, was derepressed in I363M heterozygotes, which enhanced the stemness of HSPCs. Thus, our work provides in vivo evidence that the structural integrity of EED to H3K27me3 propagation is critical, especially for embryonic development and hematopoietic homeostasis, and that its perturbation increases the predisposition to hematologic malignancies.

Maintenance of the functional integrity of mouse hematopoiesis by EED and promotion of leukemogenesis by EED haploinsufficiency.

Polycomb repressive complex 2 (PRC2) participates in transcriptional repression through methylation of histone H3K27. The WD-repeat protein embryonic ectoderm development (EED) is a non-catalytic but an essential component of PRC2 and its mutations were identified in hematopoietic malignancies. To clarify the role(s) of EED in adult hematopoiesis and leukemogenesis, we generated Eed conditional knockout (Eed(Δ/Δ)) mice. Eed(Δ/Δ) mice died in a short period with rapid decrease of hematopoietic cells. Hematopoietic stem/progenitor cells (HSPCs) were markedly decreased with impaired bone marrow (BM) repopulation ability. Cell cycle analysis of HSPCs demonstrated increased S-phase fraction coupled with suppressed G0/G1 entry. Genes encoding cell adhesion molecules are significantly enriched in Eed(Δ/Δ) HSPCs, and consistently, Eed(Δ/Δ) HSPCs exhibited increased attachment to a major extracellular matrix component, fibronectin. Thus, EED deficiency increases proliferation on one side but promotes quiescence possibly by enhanced adhesion to the hematopoietic niche on the other, and these conflicting events would lead to abnormal differentiation and functional defect of Eed(Δ/Δ) HSPCs. In addition, Eed haploinsufficiency induced hematopoietic dysplasia, and Eed heterozygous mice were susceptible to malignant transformation and developed leukemia in cooperation with Evi1 overexpression. Our results demonstrated differentiation stage-specific and dose-dependent roles of EED in normal hematopoiesis and leukemogenesis.

Fbxl10 overexpression in murine hematopoietic stem cells induces leukemia involving metabolic activation and upregulation of Nsg2.

We previously reported that deficiency for Samd9L, which was cloned as a candidate gene for -7/7q- syndrome, accelerated leukemia cooperatively with enhanced expression of a histone demethylase: F-box and leucine-rich repeat protein 10 (Fbxl10, also known as Jhdm1b, Kdm2b, and Ndy1). To further investigate the role of Fbxl10 in leukemogenesis, we generated transgenic (Tg) mice that overexpress Fbxl10 in hematopoietic stem cells (HSCs). Interestingly, Fbxl10 Tg mice developed myeloid or B-lymphoid leukemia with complete penetrance. HSCs from the Tg mice exhibited an accelerated G0/G1-to-S transition with a normal G0 to G1 entry, resulting in pleiotropic progenitor cell expansion. Fbxl10 Tg HSCs displayed enhanced expression of neuron-specific gene family member 2 (Nsg2), and forced expression of Nsg2 in primary bone marrow cells resulted in expansion of immature cells. In addition, the genes involved in mitochondrial oxidative phosphorylation were markedly enriched in Fbxl10 Tg HSCs, coupled with increased cellular adenosine 5'-triphosphate levels. Moreover, chromatin immunoprecipitation followed by sequencing analysis demonstrated that Fbxl10 directly binds to the regulatory regions of Nsg2 and oxidative phosphorylation genes. These findings define Fbxl10 as a bona fide oncogene, whose deregulated expression contributes to the development of leukemia involving metabolic proliferative advantage and Nsg2-mediated impaired differentiation.

Acquired deficiency of A20 results in rapid apoptosis, systemic inflammation, and abnormal hematopoietic stem cell function.

A20 is a negative regulator of NF-κB, and mutational loss of A20 expression is involved in the pathogenesis of autoimmune diseases and B-cell lymphomas. To clarify the role of A20 in adult hematopoiesis, we generated conditional A20 knockout mice (A20(flox/flox) ) and crossed them with Mx-1Cre (MxCre (+)) and ERT2Cre (ERT2Cre (+)) transgenic mice in which Cre is inducibly activated by endogenous interferon and exogenous tamoxifen, respectively. A20(flox/flox) MxCre (+) (A20Mx) mice spontaneously exhibited myeloid proliferation, B cell apoptosis, and anemia with overproduction of pro-inflammatory cytokines. Bone marrow transplantation demonstrated that these changes were caused by hematopoietic cells. NF-κB was constitutively activated in A20Mx hematopoietic stem cells (HSCs), which caused enhanced cell cycle entry and impaired repopulating ability. Tamoxifen stimulation of A20(flox/flox) ERT2Cre (+) (A20ERT2) mice induced fulminant apoptosis and subsequent myeloproliferation, lymphocytopenia, and progressive anemia with excessive production of pro-inflammatory cytokines, as observed in A20Mx mice. These results demonstrate that A20 plays essential roles in the homeostasis of adult hematopoiesis by preventing apoptosis and inflammation. Our findings provide insights into the mechanism underlying A20 dysfunction and human diseases in which A20 expression is impaired.

Phenylhydroquinone induces loss of thymocytes through cell cycle arrest and apoptosis elevation in p53-dependent pathway.

ortho-Phenylphenol has been employed in post-harvest treatment of citrus fruits. Although o-phenylphenol has been reported to cause carcinomas in the urinary tract in rats, toxicity to the immune organs is still unknown. Herein, we report that administration of o-phenylphenol induces thymic atrophy and loss of thymocytes in female BALB/c mice. The influence seems to result from inhibition of the thymocyte development, because increased and decreased populations of the CD4⁻ CD8⁻ double-negative and CD4⁺ CD8⁺ double-positive thymocytes were observed in the o-phenylphenol-administered mice, respectively. ortho-Phenylphenol is metabolized to phenylhydroquinone by cytochrome P450 monooxygenases. Phenylhydroquinone made cell cycle of thymocytes to be arrested through reduced expression of the genes associated with G2/M phase and through phosphorylation of p53 at Ser15. Phosphorylation of p53 at Ser15 was upregulated by activation of not only ATR but also Erk1/2 and p38, leading to increase of apoptosis. Gene expression of cytochrome P450 1A1 (CYP1A1) was promoted in thymocytes from the o-phenylphenol-administered mice. Overall, our results suggest that o-phenylphenol induces CYP1A1 expression and is metabolized into phenylhydroquinone by the expressed CYP1A1 in thymocytes. The produced phenylhydroquinone in turn induces inhibition of thymocyte development through cell cycle arrest and apoptosis in the p53-dependent pathway.