KDM4B promotes acute myeloid leukemia associated with AML1-ETO by regulating chromatin accessibility.

Epigenetic alterations of chromatin structure affect chromatin accessibility and collaborate with genetic alterations in the development of cancer. Lysine demethylase 4B (KDM4B) has been identified as a JmjC domain-containing epigenetic modifier that possesses histone demethylase activity. Although recent studies have demonstrated that KDM4B positively regulates the pathogenesis of multiple types of solid tumors, the tissue specificity and context dependency have not been fully elucidated. In this study, we investigated gene expression profiles established from clinical samples and found that KDM4B is elevated specifically in acute myeloid leukemia (AML) associated with chromosomal translocation 8;21 [t(8;21)], which results in a fusion of the AML1 and the eight-twenty-one (ETO) genes to generate a leukemia oncogene, AML1-ETO fusion transcription factor. Short hairpin RNA-mediated KDM4B silencing significantly reduced cell proliferation in t(8;21)-positive AML cell lines. Meanwhile, KDM4B silencing suppressed the expression of AML1-ETO-inducible genes, and consistently perturbed chromatin accessibility of AML1-ETO-binding sites involving altered active enhancer marks and functional cis-regulatory elements. Notably, transduction of murine KDM4B orthologue mutants followed by KDM4B silencing demonstrated a requirement of methylated-histone binding modules for a proliferative surge. To address the role of KDM4B in leukemia development, we further generated and analyzed Kdm4b conditional knockout mice. As a result, Kdm4b deficiency attenuated clonogenic potential mediated by AML1-ETO and delayed leukemia progression in vivo. Thus, our results highlight a tumor-promoting role of KDM4B in AML associated with t(8;21).

Ascorbate sensitizes human osteosarcoma cells to the cytostatic effects of cisplatin.

Osteosarcoma (OS) is the most common malignant bone tumor and a leading cause of cancer-related deaths in children and adolescents. Current standard treatments for OS are a combination of preoperative chemotherapy, surgical resection, and adjuvant chemotherapy. Cisplatin is used as the standard chemotherapeutic for OS treatment, but it induces various adverse effects, limiting its clinical application. Improving treatment efficacy without increasing the cisplatin dosage is desirable. In the present study, we assessed the combined effect of ascorbate on cisplatin treatment using cultured human OS cells. Co-treatment with ascorbate induced greater suppression of OS cell but not nonmalignant cell proliferation. The chemosensitizing effect of ascorbate on cisplatin treatment was tightly linked to ROS production. Altered cellular redox state due to increased ROS production modified glycolysis and mitochondrial function in OS cells. In addition, OS cell sphere formation was markedly decreased, suggesting that ascorbate increased the treatment efficacy of cisplatin against stem-like cells in the cancer cell population. We also found that enhanced MYC signaling, ribosomal biogenesis, glycolysis, and mitochondrial respiration are key signatures in OS cells with cisplatin resistance. Furthermore, cisplatin resistance was reversed by ascorbate. Taken together, our findings provide a rationale for combining cisplatin with ascorbate in therapeutic strategies against OS.

High Fat Diet Triggers a Reduction in Body Fat Mass in Female Mice Deficient for Utx demethylase.

Obesity increases the risk of metabolic disorders like diabetes mellitus and dyslipidemia. However, how metabolic status is sensed and regulates cellular behavior is unclear. Utx is an H3K27 demethylase that influences adipocyte function in vitro. To examine its role in vivo, we generated mice lacking Utx in adipocytes (UtxAKO). Although all UtxAKO mice grew normally on a normal chow diet (NCD), female UtxAKO mice on a high fat diet (HFD) showed striking reductions in body fat compared to control mice (Ctrl). Gene expression profiling of adipose tissues of HFD-fed UtxAKO female mice revealed decreased expression of rate-limiting enzymes of triacylglycerol synthesis but increased expression of those of cholesterol/steroid hormone synthesis. Moreover, these animals resisted adiposity induced by ovariectomy and exhibited increased estrogen in visceral adipose tissues. Thus, upon HFD feeding, Utx regulates lipid metabolism in adipose tissues by influencing the local hormonal microenvironment. Conversely, Utx deficiency skews lipid catabolism to enhance cholesterol/steroid hormone production and repress obesity.

JMJD2B/KDM4B inactivation in adipose tissues accelerates obesity and systemic metabolic abnormalities.

Obesity is a serious global health issue; however, the roles of genetics and epigenetics in the onset and progression of obesity are still not completely understood. The aim of this study was to determine the role of Kdm4b, which belongs to a subfamily of histone demethylases, in adipogenesis and fat metabolism in vivo. We established conditional Kdm4b knockout mice. Inactivation of Kdm4b in adipocytes (K4bKO) induced profound obesity in mice on a high fat diet (HFD). The HFD-fed K4bKO mice exhibited an increased volume of fat mass and higher expression levels of adipogenesis-related genes. In contrast, the genes involved in energy expenditure and mitochondrial functions were down-regulated. Supporting these findings, the energy expenditure of Kdm4b-deficient cells was markedly decreased. In addition, progression of glucose intolerance and hepatic steatosis with hepatocellular damages was observed. These data indicate that Kdm4b is a critical regulator of systemic metabolism via enhancing energy expenditure in adipocytes.

The H3K27 demethylase, Utx, regulates adipogenesis in a differentiation stage-dependent manner.

Understanding the molecular mechanisms that drive adipogenesis is important in developing new treatments for obesity and diabetes. Epigenetic regulations determine the capacity of adipogenesis. In this study, we examined the role of a histone H3 lysine 27 demethylase, the ubiquitously transcribed tetratricopeptide repeat protein on the X chromosome (Utx), in the differentiation of mouse embryonic stem cells (mESCs) to adipocytes. Using gene trapping, we examined Utx-deficient male mESCs to determine whether loss of Utx would enhance or inhibit the differentiation of mESCs to adipocytes. Utx-deficient mESCs showed diminished potential to differentiate to adipocytes compared to that of controls. In contrast, Utx-deficient preadipocytes showed enhanced differentiation to adipocytes. Microarray analyses indicated that the ß-catenin/c-Myc signaling pathway was differentially regulated in Utx-deficient cells during adipocyte differentiation. Therefore, our data suggest that Utx governs adipogenesis by regulating c-Myc in a differentiation stage-specific manner and that targeting the Utx signaling pathway could be beneficial for the treatment of obesity, diabetes, and congenital utx-deficiency disorders.

Attenuated DNA damage repair delays therapy-related myeloid neoplasms in a mouse model.

Therapy-related cancers are potentially fatal late life complications for patients who received radio- or chemotherapy. So far, the mouse model showing reduction or delay of these diseases has not been described. We found that the disruption of Aplf in mice moderately attenuated DNA damage repair and, unexpectedly, impeded myeloid neoplasms after exposure to ionizing radiation (IR). Irradiated mutant mice showed higher rates of p53-dependent cell death, fewer chromosomal translocations, and a delay in malignancy-induced mortality. Simultaneous deficiency of p53 abrogated IR-induced apoptosis and the benefit of impaired DNA repair on mortality in irradiated Aplf­/­ mice. Depletion of APLF in non-tumorigenic human cells also markedly reduced the risk of radiation-induced chromosomal aberrations. We therefore conclude that proficient DNA damage repair may promote chromosomal aberrations in normal tissues after irradiation and induce malignant evolution, thus illustrating the potential benefit in sensitizing p53 function by manipulating DNA repair efficiency in cancer patients undergoing genotoxic therapies.

Bach1 deficiency and accompanying overexpression of heme oxygenase-1 do not influence aging or tumorigenesis in mice.

Oxidative stress contributes to both aging and tumorigenesis. The transcription factor Bach1, a regulator of oxidative stress response, augments oxidative stress by repressing the expression of heme oxygenase-1 (HO-1) gene (Hmox1) and suppresses oxidative stress-induced cellular senescence by restricting the p53 transcriptional activity. Here we investigated the lifelong effects of Bach1 deficiency on mice. Bach1-deficient mice showed longevity similar to wild-type mice. Although HO-1 was upregulated in the cells of Bach1-deficient animals, the levels of ROS in Bach1-deficient HSCs were comparable to those in wild-type cells. Bach1(-/-); p53(-/-) mice succumbed to spontaneous cancers as frequently as p53-deficient mice. Bach1 deficiency significantly altered transcriptome in the liver of the young mice, which surprisingly became similar to that of wild-type mice during the course of aging. The transcriptome adaptation to Bach1 deficiency may reflect how oxidative stress response is tuned upon genetic and environmental perturbations. We concluded that Bach1 deficiency and accompanying overexpression of HO-1 did not influence aging or p53 deficiency-driven tumorigenesis. Our results suggest that it is useful to target Bach1 for acute injury responses without inducing any apparent deteriorative effect.

Bach1-mediated suppression of p53 is inhibited by p19(ARF) independently of MDM2.

Cellular senescence prevents the aberrant proliferation of damaged cells. The transcription factor Bach1 binds to p53 to repress cellular senescence, but it is still unclear how the Bach1-p53 interaction is regulated. We found that the Bach1-p53 interaction was inhibited by oncogenic Ras, bleomycin, and hydrogen peroxide. Proteomics analysis of Bach1 complex revealed its interaction with p19(ARF), a tumor suppressor that competitively inhibited the Bach1-p53 interaction when overexpressed within cells. Reduction of MDM2 expression in wild-type murine embryonic fibroblasts (MEFs) did not result in slower proliferation, showing that Bach1 plays a role in keeping the proliferation of MEFs independent of MDM2. Consistent with this interpretation, expression of p21 was highly induced in MEFs when both Bach1 and MDM2 were abrogated. The level of Bach1 protein was reduced on knockdown of p53. These results suggest that p53 activation involves its dissociation from Bach1, achieved in part by the competitive binding of p19(ARF) to Bach1. The p19(ARF)-Bach1 interaction constitutes a regulatory pathway of p53 in parallel with the p19(ARF)-MDM2 pathway.

Identification of senescence-associated genes and their networks under oxidative stress by the analysis of Bach1.

Cellular senescence is induced in response to DNA damage, caused by genotoxic stresses, including oxidative stress, and serves as a barrier against malignant transformation. Tumor-suppressor protein p53 induces genes critical for implementing cellular senescence. However, the identities of p53 target genes and other regulators that achieve senescence under oxidative stress remain to be elucidated. Effector genes for oxidative stress-induced cellular senescence were sought, based on the fact that transcription factor Bach1 inhibits this response by impeding the transcriptional activity of p53. pRb became hypophosphorylated more rapidly in Bach1-deficient MEFs than in wild-type cells, suggesting that pRb activation was involved in their senescence. Bach1-deficient MEFs bypassed the senescence state when the expression of a subset of p53 target genes, including p21, Pai1, Noxa, and Perp, was simultaneously reduced by using RNAi. Combined knockdown of p21 and pRb resulted in vigorous re-proliferation. These results suggest that oxidative stress-induced cellular senescence is registered by multiple p53 target genes, which arrest proliferation redundantly, in part by activating pRb. Our elucidations contrast with previous reports describing monopolistic regulations of senescence by single p53 target genes.

[Regulation of cellular senescence by Bach1].

The tumor suppressor p53 induces cellular senescence, an irreversible form of proliferation arrest, to inhibit carcinogenesis as well as aging of organs and a body. While the major cause of cellular senescence and aging is oxidative stress, little is known about how the p53 activity is regulated under such conditions. Bach1 inhibits expression of oxidative stress responsive genes by competing with Nrf2, the key activator of oxidative stress response. Bach1 inhibits p53-dependent cellular senescence induced by oxidative stress. Bach1 forms a protein complex with p53, is recruited to p53 target genes, and inhibits their expression. These findings provide completely novel insights into how the activity of p53 is regulated under oxidative stress. Since p53 is the critical tumor suppressor with huge clinical implications, the newly identified mechanism should open new research fields.

Bach1 inhibits oxidative stress-induced cellular senescence by impeding p53 function on chromatin.

Cellular senescence is one of the key strategies to suppress expansion of cells with mutations. Senescence is induced in response to genotoxic and oxidative stress. Here we show that the transcription factor Bach1 (BTB and CNC homology 1, basic leucine zipper transcription factor 1), which inhibits oxidative stress-inducible genes, is a crucial negative regulator of oxidative stress-induced cellular senescence. Bach1-deficient murine embryonic fibroblasts showed a propensity to undergo more rapid and profound p53-dependent premature senescence than control wild-type cells in response to oxidative stress. Bach1 formed a complex that contained p53, histone deacetylase 1 and nuclear co-repressor N-coR. Bach1 was recruited to a subset of p53 target genes and contributed to impeding p53 action by promoting histone deacetylation. Because Bach1 is regulated by oxidative stress and heme, our data show that Bach1 connects oxygen metabolism and cellular senescence as a negative regulator of p53.