PRC2 Inhibitors Overcome Glucocorticoid Resistance Driven by NSD2 Mutation in Pediatric Acute Lymphoblastic Leukemia.

Mutations in epigenetic regulators are common in relapsed pediatric acute lymphoblastic leukemia (ALL). Here, we uncovered the mechanism underlying the relapse of ALL driven by an activating mutation of the NSD2 histone methyltransferase (p.E1099K). Using high-throughput drug screening, we found that NSD2-mutant cells were specifically resistant to glucocorticoids. Correction of this mutation restored glucocorticoid sensitivity. The transcriptional response to glucocorticoids was blocked in NSD2-mutant cells due to depressed glucocorticoid receptor (GR) levels and the failure of glucocorticoids to autoactivate GR expression. Although H3K27me3 was globally decreased by NSD2 p.E1099K, H3K27me3 accumulated at the NR3C1 (GR) promoter. Pretreatment of NSD2 p.E1099K cell lines and patient-derived xenograft samples with PRC2 inhibitors reversed glucocorticoid resistance in vitro and in vivo. PRC2 inhibitors restored NR3C1 autoactivation by glucocorticoids, increasing GR levels and allowing GR binding and activation of proapoptotic genes. These findings suggest a new therapeutic approach to relapsed ALL associated with NSD2 mutation. SIGNIFICANCE: NSD2 histone methyltransferase mutations observed in relapsed pediatric ALL drove glucocorticoid resistance by repression of the GR and abrogation of GR gene autoactivation due to accumulation of K3K27me3 at its promoter. Pretreatment with PRC2 inhibitors reversed resistance, suggesting a new therapeutic approach to these patients with ALL.This article is highlighted in the In This Issue feature, p. 1.

An activating mutation of the NSD2 histone methyltransferase drives oncogenic reprogramming in acute lymphocytic leukemia.

NSD2, a histone methyltransferase specific for methylation of histone 3 lysine 36 (H3K36), exhibits a glutamic acid to lysine mutation at residue 1099 (E1099K) in childhood acute lymphocytic leukemia (ALL), and cells harboring this mutation can become the predominant clone in relapsing disease. We studied the effects of this mutant enzyme in silico, in vitro, and in vivo using gene edited cell lines. The E1099K mutation altered enzyme/substrate binding and enhanced the rate of H3K36 methylation. As a result, cell lines harboring E1099K exhibit increased H3K36 dimethylation and reduced H3K27 trimethylation, particularly on nucleosomes containing histone H3.1. Mutant NSD2 cells exhibit reduced apoptosis and enhanced proliferation, clonogenicity, adhesion, and migration. In mouse xenografts, mutant NSD2 cells are more lethal and brain invasive than wildtype cells. Transcriptional profiling demonstrates that mutant NSD2 aberrantly activates factors commonly associated with neural and stromal lineages in addition to signaling and adhesion genes. Identification of these pathways provides new avenues for therapeutic interventions in NSD2 dysregulated malignancies.

The Role of Nuclear Receptor-Binding SET Domain Family Histone Lysine Methyltransferases in Cancer.

The nuclear receptor-binding SET Domain (NSD) family of histone H3 lysine 36 methyltransferases is comprised of NSD1, NSD2 (MMSET/WHSC1), and NSD3 (WHSC1L1). These enzymes recognize and catalyze methylation of histone lysine marks to regulate chromatin integrity and gene expression. The growing number of reports demonstrating that alterations or translocations of these genes fundamentally affect cell growth and differentiation leading to developmental defects illustrates the importance of this family. In addition, overexpression, gain of function somatic mutations, and translocations of NSDs are associated with human cancer and can trigger cellular transformation in model systems. Here we review the functions of NSD family members and the accumulating evidence that these proteins play key roles in tumorigenesis. Because epigenetic therapy is an important emerging anticancer strategy, understanding the function of NSD family members may lead to the development of novel therapies.

Sabotaging of the oxidative stress response by an oncogenic noncoding RNA.

Overexpression of the multiple myeloma set domain (MMSET) Wolf-Hirschhorn syndrome candidate 1 gene, which contains an orphan box H/ACA class small nucleolar RNA, ACA11, in an intron, is associated with several cancer types, including multiple myeloma (MM). ACA11 and MMSET are overexpressed cotranscriptionally as a result of the t(4;14) chromosomal translocation in a subset of patients with MM. RNA sequencing of CD138+ tumor cells from t(4;14)-positive and -negative MM patient bone marrow samples revealed an enhanced oxidative phosphorylation mRNA signature. Supporting these data, ACA11 overexpression in a t(4;14)-negative MM cell line, MM1.S, demonstrated enhanced reactive oxygen species (ROS) levels. In addition, an enhancement of cell proliferation, increased soft agar colony size, and elevated ERK1/2 phosphorylation were observed. This ACA11-driven hyperproliferative phenotype depended on increased ROS levels as exogenously added antioxidants attenuate the increased proliferation. A major transcriptional regulator of the cellular antioxidant response, nuclear factor (erythroid-derived 2)-like 2 (NRF2), shuttled to the nucleus, as expected, in response to ACA11-driven increases in ROS; however, transcriptional up-regulation of some of NRF2's antioxidant target genes was abrogated in the presence of ACA11 overexpression. These data show for the first time that ACA11 promotes proliferation through inhibition of NRF2 function resulting in sustained ROS levels driving cancer cell proliferation.-Mahajan, N., Wu, H.-J., Bennett, R. L., Troche, C., Licht, J. D., Weber, J. D., Maggi, L. B., Jr., Tomasson, M. H. Sabotaging of the oxidative stress response by an oncogenic noncoding RNA.

Hepatic ZIP14-mediated Zinc Transport Contributes to Endosomal Insulin Receptor Trafficking and Glucose Metabolism.

Zinc influences signaling pathways through controlled targeted zinc transport. Zinc transporter Zip14 KO mice display a phenotype that includes impaired intestinal barrier function with low grade chronic inflammation, hyperinsulinemia, and increased body fat, which are signatures of diet-induced diabetes (type 2 diabetes) and obesity in humans. Hyperglycemia in type 2 diabetes and obesity is caused by insulin resistance. Insulin resistance results in inhibition of glucose uptake by liver and other peripheral tissues, principally adipose and muscle and with concurrently higher hepatic glucose production. Therefore, modulation of hepatic glucose metabolism is an important target for antidiabetic treatment approaches. We demonstrate that during glucose uptake, cell surface abundance of zinc transporter ZIP14 and mediated zinc transport increases. Zinc is distributed to multiple sites in hepatocytes through sequential translocation of ZIP14 from plasma membrane to early and late endosomes. Endosomes from Zip14 KO mice were zinc-deficient because activities of the zinc-dependent insulin-degrading proteases insulin-degrading enzyme and cathepsin D were impaired; hence insulin receptor activity increased. Transient increases in cytosolic zinc levels are concurrent with glucose uptake and suppression of glycogen synthesis. In contrast, Zip14 KO mice exhibited greater hepatic glycogen synthesis and impaired gluconeogenesis and glycolysis related to low cytosolic zinc levels. We can conclude that ZIP14-mediated zinc transport contributes to regulation of endosomal insulin receptor activity and glucose homeostasis in hepatocytes. Therefore, modulation of ZIP14 transport activity presents a new target for management of diabetes and other glucose-related disorders.

Aging amplifies multiple phenotypic defects in mice with zinc transporter Zip14 (Slc39a14) deletion.

Inflammation and zinc dyshomeostasis are two common hallmarks of aging. A major zinc transporter ZIP14 (slc39a14) is upregulated by proinflammatory stimuli, e.g. interleukin-6. We have evaluated the influence of age on the Zip14 KO phenotype using wild-type (WT) and Zip14 knockout (KO) mice. Aging produced a major increase in serum IL-6 concentrations that was dramatically augmented in the Zip14 KO mice. In keeping with enhanced serum IL-6 concentrations, aging produced tissue-specific increases in zinc concentration of skeletal muscle and white adipose tissue. Metabolic endotoxemia produced by Zip14 ablation is maintained in aged KO mice. Muscle non-heme iron (NHI) was increased in aged WT mice but not in aged Zip14 KO mice demonstrating NHI uptake by muscle is ZIP14-dependent and increases with age. NF-κB and STAT3 activation was greater in aged mice, but was tissue specific and inversely related to tissue zinc. Micro-CT analysis revealed that Zip14 KO mice had markedly reduced trabecular bone that was greatly amplified with aging. These results demonstrate that the inflammation-responsive zinc transporter ZIP14 has phenotypic effects that are amplified with aging.

Zinc transporter Slc39a14 regulates inflammatory signaling associated with hypertrophic adiposity.

Zinc is a signaling molecule in numerous metabolic pathways, the coordination of which occurs through activity of zinc transporters. The expression of zinc transporter Zip14 (Slc39a14), a zinc importer of the solute carrier 39 family, is stimulated under proinflammatory conditions. Adipose tissue upregulates Zip14 during lipopolysaccharide-induced endotoxemia. A null mutation of Zip14 (KO) revealed that phenotypic changes in adipose include increased cytokine production, increased plasma leptin, hypertrophied adipocytes, and dampened insulin signaling. Adipose tissue from KO mice had increased levels of preadipocyte markers, lower expression of the differentiation marker (PPARγ), and activation of NF-κB and STAT3 pathways. Our overall hypothesis was that ZIP14 would play a role in adipocyte differentiation and inflammatory obesity. Global Zip14 KO causes systemic endotoxemia. The observed metabolic changes in adipose metabolism were reversed when oral antibiotics were administrated, indicating that circulating levels of endotoxin were in part responsible for the adipose phenotype. To evaluate a mechanism, 3T3-L1 cells were differentiated into adipocytes and treated with siRNA to knock down Zip14. These cells had an impaired ability to mobilize zinc, which caused dysregulation of inflammatory pathways (JAK2/STAT3 and NF-κB). The Zip14 deletion may limit the availability of intracellular zinc, yielding the unique phenotype of inflammation coupled with hypertrophy. Taken together, these results suggest that aberrant zinc distribution observed with Zip14 ablation impacts adipose cytokine production and metabolism, ultimately increasing fat deposition when exposed to endotoxin. To our knowledge, this is the first investigation into the mechanistic role of ZIP14 in adipose tissue regulation and metabolism.

The influence of dietary zinc source and coccidial vaccine exposure on intracellular zinc homeostasis and immune status in broiler chickens.

Coccidia are protozoal parasites which compromise mucosal integrity of the intestine, potentiating poultry morbidity. The host's Zn status influences the course of infection. Therefore, two experiments were designed to determine how supplemental Zn regimens impacted jejunal and caecal immune status and Zn transporter expression. Coccivac®-B was administered weekly at ten times the recommended dose as a mild coccidial challenge (10 CV). Zn was provided through a basal diet, supplemental zinc sulfate (ZnSO4), or a supplemental 1:1 blend of ZnSO4 and Availa®-Zn (Blend). Mucosal jejunum (Expt 1) and caecal tonsils (Expt 2) were evaluated for intracellular Zn concentrations and phagocytic capacity. Messenger expression of Zn transporters ZnT5, ZnT7, Zip9 and Zip13 were investigated to determine Zn trafficking. With 10 CV, phagocytic capacity was decreased in jejunal cells by 2%. In the caecal tonsils, however, phagocytic capacity increased with challenge, with the magnitude of increase being more pronounced with higher dietary Zn (10 CV × Zn interaction; P = 0.04). Intracellular Zn within caecal tonsils was found significantly reduced with 10 CV (27%, P = 0.0001). 10 CV also resulted in an overall increase in the ratio of Zip:ZnT transporters. With the exception of Zip13 transporter expression, dietary Zn source had little impact on any of the measured cellular parameters. Thus, intestinal mucosal tissues had reductions in intracellular free Zn during coccidial challenge, which was coupled with an upregulation of measured Zip transporters. This suggests that under coccidial challenge, intestinal cells attempt to compensate for the drop in intracellular Zn.

Influence of ZIP14 (slc39A14) on intestinal zinc processing and barrier function.

ZIP14 is a zinc transport protein with high expression in the small intestine and liver. Zip14 is upregulated during endotoxemia and leads to increased liver zinc content and transient hypozinemia. Since body zinc status and inflammation are associated with changes in intestinal permeability, we hypothesized that ZIP14 may influence intestinal permeability. Wild-type (WT) and Zip14 knockout (KO) mice were used to determine ZIP14-associated intestinal zinc metabolism and effects on permeability. Fractionation of plasma membranes revealed that ZIP14 is localized to the basolateral membrane of enterocytes. Studies utilizing (65)Zn administered by subcutaneous injection revealed greater zinc accumulation in the SI of Zip14 KO mice compared with WT mice. Isolation of endosomes confirmed the presence of ZIP14. Quantification of endosomal zinc concentration by FluoZin-3AM fluorescence demonstrated that zinc is trapped in endosomes of Zip14 KO mice. Intestinal permeability assessed both by plasma FITC-dextran following gavage and by serum endotoxin content was greater in Zip14 KO mice. Threonine phosphorylation of the tight junction protein occludin, which is necessary for tight junction assembly, was reduced in KO mice. Claudin 1 and 2, known to have an inverse relationship in regards to tight junction integrity, reflected impaired barrier function in KO jejunum. These data suggest involvement of ZIP14 in providing zinc for a regulatory role needed for maintenance of the intestinal barrier. In conclusion, ZIP14 is a basolaterally localized protein in enterocytes and is involved in endosomal trafficking of zinc and is necessary for proper maintenance of intestinal tight junctions.