Small-molecule inhibitors targeting Polycomb repressive complex 1 RING domain.

Polycomb repressive complex 1 (PRC1) is an essential chromatin-modifying complex that monoubiquitinates histone H2A and is involved in maintaining the repressed chromatin state. Emerging evidence suggests PRC1 activity in various cancers, rationalizing the need for small-molecule inhibitors with well-defined mechanisms of action. Here, we describe the development of compounds that directly bind to RING1B-BMI1, the heterodimeric complex constituting the E3 ligase activity of PRC1. These compounds block the association of RING1B-BMI1 with chromatin and inhibit H2A ubiquitination. Structural studies demonstrate that these inhibitors bind to RING1B by inducing the formation of a hydrophobic pocket in the RING domain. Our PRC1 inhibitor, RB-3, decreases the global level of H2A ubiquitination and induces differentiation in leukemia cell lines and primary acute myeloid leukemia (AML) samples. In summary, we demonstrate that targeting the PRC1 RING domain with small molecules is feasible, and RB-3 represents a valuable chemical tool to study PRC1 biology.

Covalent inhibition of NSD1 histone methyltransferase.

The nuclear receptor-binding SET domain (NSD) family of histone methyltransferases is associated with various malignancies, including aggressive acute leukemia with NUP98-NSD1 translocation. While NSD proteins represent attractive drug targets, their catalytic SET domains exist in autoinhibited conformation, presenting notable challenges for inhibitor development. Here, we employed a fragment-based screening strategy followed by chemical optimization, which resulted in the development of the first-in-class irreversible small-molecule inhibitors of the nuclear receptor-binding SET domain protein 1 (NSD1) SET domain. The crystal structure of NSD1 in complex with covalently bound ligand reveals a conformational change in the autoinhibitory loop of the SET domain and formation of a channel-like pocket suitable for targeting with small molecules. Our covalent lead-compound BT5-demonstrates on-target activity in NUP98-NSD1 leukemia cells, including inhibition of histone H3 lysine 36 dimethylation and downregulation of target genes, and impaired colony formation in an NUP98-NSD1 patient sample. This study will facilitate the development of the next generation of potent and selective inhibitors of the NSD histone methyltransferases.

C/EBPα is an essential collaborator in Hoxa9/Meis1-mediated leukemogenesis.

Homeobox A9 (HOXA9) is a homeodomain-containing transcription factor that plays a key role in hematopoietic stem cell expansion and is commonly deregulated in human acute leukemias. A variety of upstream genetic alterations in acute myeloid leukemia (AML) lead to overexpression of HOXA9, almost always in association with overexpression of its cofactor meis homeobox 1 (MEIS1) . A wide range of data suggests that HOXA9 and MEIS1 play a synergistic causative role in AML, although the molecular mechanisms leading to transformation by HOXA9 and MEIS1 remain elusive. In this study, we identify CCAAT/enhancer binding protein alpha (C/EBPα) as a critical collaborator required for Hoxa9/Meis1-mediated leukemogenesis. We show that C/EBPα is required for the proliferation of Hoxa9/Meis1-transformed cells in culture and that loss of C/EBPα greatly improves survival in both primary and secondary murine models of Hoxa9/Meis1-induced leukemia. Over 50% of Hoxa9 genome-wide binding sites are cobound by C/EBPα, which coregulates a number of downstream target genes involved in the regulation of cell proliferation and differentiation. Finally, we show that Hoxa9 represses the locus of the cyclin-dependent kinase inhibitors Cdkn2a/b in concert with C/EBPα to overcome a block in G1 cell cycle progression. Together, our results suggest a previously unidentified role for C/EBPα in maintaining the proliferation required for Hoxa9/Meis1-mediated leukemogenesis.

The same site on the integrase-binding domain of lens epithelium-derived growth factor is a therapeutic target for MLL leukemia and HIV.

Lens epithelium-derived growth factor (LEDGF) is a chromatin-associated protein implicated in leukemia and HIV type 1 infection. LEDGF associates with mixed-lineage leukemia (MLL) fusion proteins and menin and is required for leukemic transformation. To better understand the molecular mechanism underlying the LEDGF integrase-binding domain (IBD) interaction with MLL fusion proteins in leukemia, we determined the solution structure of the MLL-IBD complex. We found a novel MLL motif, integrase domain binding motif 2 (IBM2), which binds to a well-defined site on IBD. Point mutations within IBM2 abolished leukemogenic transformation by MLL-AF9, validating that this newly identified motif is essential for the oncogenic activity of MLL fusion proteins. Interestingly, the IBM2 binding site on IBD overlaps with the binding site for the HIV integrase (IN), and IN was capable of efficiently sequestering IBD from the menin-MLL complex. A short IBM2 peptide binds to IBD directly and inhibits both the IBD-MLL/menin and IBD-IN interactions. Our findings show that the same site on IBD is involved in binding to MLL and HIV-IN, revealing an attractive approach to simultaneously target LEDGF in leukemia and HIV.

SRA gene knockout protects against diet-induced obesity and improves glucose tolerance.

We have recently shown that the non-coding RNA, steroid receptor RNA activator (SRA), functions as a transcriptional coactivator of PPARγ and promotes adipocyte differentiation in vitro. To assess SRA function in vivo, we have generated a whole mouse Sra1 gene knock-out (SRA(-/-)). Here, we show that the Sra1 gene is an important regulator of adipose tissue mass and function. SRA is expressed at a higher level in adipose tissue than other organs in wild type mice. SRA(-/-) mice are resistant to high fat diet-induced obesity, with decreased fat mass and increased lean content. This lean phenotype of SRA(-/-) mice is associated with decreased expression of a subset of adipocyte marker genes and reduced plasma TNFα levels. The SRA(-/-) mice are more insulin sensitive, as evidenced by reduced fasting insulin, and lower blood glucoses in response to IP glucose and insulin. In addition, the livers of SRA(-/-) mice have fewer lipid droplets after high fat diet feeding, and the expression of lipogenesis-associated genes is decreased. To our knowledge, these data are the first to indicate a functional role for SRA in adipose tissue biology and glucose homeostasis in vivo.

The unfolded protein response transducer IRE1α prevents ER stress-induced hepatic steatosis.

The endoplasmic reticulum (ER) is the cellular organelle responsible for protein folding and assembly, lipid and sterol biosynthesis, and calcium storage. The unfolded protein response (UPR) is an adaptive intracellular stress response to accumulation of unfolded or misfolded proteins in the ER. In this study, we show that the most conserved UPR sensor inositol-requiring enzyme 1 α (IRE1α), an ER transmembrane protein kinase/endoribonuclease, is required to maintain hepatic lipid homeostasis under ER stress conditions through repressing hepatic lipid accumulation and maintaining lipoprotein secretion. To elucidate physiological roles of IRE1α-mediated signalling in the liver, we generated hepatocyte-specific Ire1α-null mice by utilizing an albumin promoter-controlled Cre recombinase-mediated deletion. Deletion of Ire1α caused defective induction of genes encoding functions in ER-to-Golgi protein transport, oxidative protein folding, and ER-associated degradation (ERAD) of misfolded proteins, and led to selective induction of pro-apoptotic UPR trans-activators. We show that IRE1α is required to maintain the secretion efficiency of selective proteins. In the absence of ER stress, mice with hepatocyte-specific Ire1α deletion displayed modest hepatosteatosis that became profound after induction of ER stress. Further investigation revealed that IRE1α represses expression of key metabolic transcriptional regulators, including CCAAT/enhancer-binding protein (C/EBP) ß, C/EBPδ, peroxisome proliferator-activated receptor γ (PPARγ), and enzymes involved in triglyceride biosynthesis. IRE1α was also found to be required for efficient secretion of apolipoproteins upon disruption of ER homeostasis. Consistent with a role for IRE1α in preventing intracellular lipid accumulation, mice with hepatocyte-specific deletion of Ire1α developed severe hepatic steatosis after treatment with an ER stress-inducing anti-cancer drug Bortezomib, upon expression of a misfolding-prone human blood clotting factor VIII, or after partial hepatectomy. The identification of IRE1α as a key regulator to prevent hepatic steatosis provides novel insights into ER stress mechanisms in fatty liver diseases associated with toxic liver injuries.

Conservative mutations in the C2 domains of factor VIII and factor V alter phospholipid binding and cofactor activity.

Factor VIII and factor V share structural homology and bind to phospholipid membranes via tandem, lectin-like C domains. Their respective C2 domains bind via 2 pairs of hydrophobic amino acids and an amphipathic cluster. In contrast, the factor V-like, homologous subunit (Pt-FV) of a prothrombin activator from Pseudonaja textilis venom is reported to function without membrane binding. We hypothesized that the distinct membrane-interactive amino acids of these proteins contribute to the differing membrane-dependent properties. We prepared mutants in which the C2 domain hydrophobic amino acid pairs were changed to the homologous residues of the other protein and a factor V mutant with 5 amino acids changed to those from Pt-FV (FV(MTTS/Y)). Factor VIII mutants were active on additional membrane sites and had altered apparent affinities for factor X. Some factor V mutants, including FV(MTTS/Y), had increased membrane interaction and apparent membrane-independent activity that was the result of phospholipid retained during purification. Phospholipid-free FV(MTTS/Y) showed increased activity, particularly a 10-fold increase in activity on membranes lacking phosphatidylserine. The reduced phosphatidylserine requirement correlated to increased activity on resting and stimulated platelets. We hypothesize that altered membrane binding contributes to toxicity of Pt-FV.

Combination of menin and kinase inhibitors as an effective treatment for leukemia with NUP98 translocations.

Chromosomal translocations of the nucleoporin 98 (NUP98) gene are found in acute myeloid leukemia (AML) patients leading to very poor outcomes. The oncogenic activity of NUP98 fusion proteins is dependent on the interaction between Mixed Lineage Leukemia 1 and menin. NUP98-rearranged (NUP98-r) leukemia cells also rely on specific kinases, including CDK6 and/or FLT3, suggesting that simultaneous targeting of these kinases and menin could overcome limited sensitivity to single agents. Here, we found that combinations of menin inhibitor, MI-3454, with kinase inhibitors targeting either CDK6 (Palbociclib) or FLT3 (Gilteritinib) strongly enhance the anti-leukemic effect of menin inhibition in NUP98-r leukemia models. We found strong synergistic effects of both combinations on cell growth, colony formation and differentiation in patient samples with NUP98 translocations. These combinations also markedly augmented anti-leukemic efficacy of menin inhibitor in Patient Derived Xenograft models of NUP98-r leukemia. Despite inhibiting two unrelated kinases, when Palbociclib or Gilteritinib were combined with the menin inhibitor, they affected similar pathways relevant to leukemogenesis, including cell cycle regulation, cell proliferation and differentiation. This study provides strong rationale for clinical translation of the combination of menin and kinase inhibitors as novel treatments for NUP98-r leukemia, supporting the unexplored combinations of epigenetic drugs with kinase inhibitors.

A membrane-interactive surface on the factor VIII C1 domain cooperates with the C2 domain for cofactor function.

Factor VIII binds to phosphatidylserine (PS)-containing membranes through its tandem, lectin-homology, C1 and C2 domains. However, the details of C1 domain membrane binding have not been delineated. We prepared 4 factor VIII C1 mutations localized to a hypothesized membrane-interactive surface (Arg2090Ala/Gln2091Ala, Lys2092Ala/Phe2093Ala, Gln2042Ala/Tyr2043Ala, and Arg2159Ala). Membrane binding and cofactor activity were measured using membranes with 15% PS, mimicking platelets stimulated by thrombin plus collagen, and 4% PS, mimicking platelets stimulated by thrombin. All mutants had at least 10-fold reduced affinities for membranes of 4% PS, and 3 mutants also had decreased apparent affinity for factor X. Monoclonal antibodies against the C2 domain produced different relative impairment of mutants compared with wild-type factor VIII. Monoclonal antibody ESH4 decreased the V(max) for all mutants but only the apparent membrane affinity for wild-type factor VIII. Monoclonal antibody BO2C11 decreased the V(max) of wild-type factor VIII by 90% but decreased the activity of 3 mutants more than 98%. These results identify a membrane-binding face of the factor VIII C1 domain, indicate an influence of the C1 domain on factor VIII binding to factor X, and indicate that cooperation between the C1 and C2 domains is necessary for full activity of the factor Xase complex.

[Role of MEK1/ERK1, 2 pathways in the calcium-sensing receptor mediation of hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells].

OBJECTIVE: To explore the cell signal transduction pathway of calcium-sensing receptor (CaSR) mediated hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells (PASMCs). METHODS: The expressions of proliferating cell nuclear antigen (PCNA) and extracellular signal-regulated protein kinase 1, 2 (ERK1, 2) were analyzed by Western blot. Cell proliferation was tested by a 5-bromo-2-deoxyuridine (BrdU) incorporation assay. Cell cycle and proliferation index (PI) were analyzed by flow cytometry. RESULTS: Hypoxia significantly increased the expression of PCNA (0.528 ± 0.028), p-ERK1, 2 (1.12 ± 0.05, 0.91 ± 0.06), BrdU incorporation (143.3 ± 4.2) and cell proliferation index (12.5 ± 0.9) (all P < 0.05, versus control group, 0.243 ± 0.025, 0.47 ± 0.03, 0.40 ± 0.03, 100.0 ± 5.4, 7.5 ± 1.2). Gadolinium chloride (GdCl3, a CaSR agonist) amplified the effect of hypoxia (0.770 ± 0.039, 1.50 ± 0.06, 1.61 ± 0.05, 187.4 ± 3.9, 19.8 ± 0.6, all P < 0.05). PD98059 (a MEK1 inhibitor) decreased the up-regulation of PCNA expression, BrdU incorporation and the increase of cell proliferation index induced by hypoxia and GdCl3 in PASMCs (0.441 ± 0.020, 0.71 ± 0.07, 0.72 ± 0.06, 115.5 ± 4.0, 9.3 ± 1.1, all P < 0.05). CONCLUSION: Calcium-sensing receptor mediates hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells through ERK1, 2 pathways.

Mutant NPM1 Hijacks Transcriptional Hubs to Maintain Pathogenic Gene Programs in Acute Myeloid Leukemia.

Nucleophosmin (NPM1) is a ubiquitously expressed nucleolar protein with a wide range of biological functions. In 30% of acute myeloid leukemia (AML), the terminal exon of NPM1 is often found mutated, resulting in the addition of a nuclear export signal and a shift of the protein to the cytoplasm (NPM1c). AMLs carrying this mutation have aberrant expression of the HOXA/B genes, whose overexpression leads to leukemogenic transformation. Here, for the first time, we comprehensively prove that NPM1c binds to a subset of active gene promoters in NPM1c AMLs, including well-known leukemia-driving genes-HOXA/B cluster genes and MEIS1. NPM1c sustains the active transcription of key target genes by orchestrating a transcription hub and maintains the active chromatin landscape by inhibiting the activity of histone deacetylases. Together, these findings reveal the neomorphic function of NPM1c as a transcriptional amplifier for leukemic gene expression and open up new paradigms for therapeutic intervention. SIGNIFICANCE: NPM1 mutation is the most common mutation in AML, yet the mechanism of how the mutant protein results in AML remains unclear. Here, for the first time, we prove mutant NPM1 directly binds to active chromatin regions and hijacks the transcription of AML-driving genes. See related article by Uckelmann et al., p. 746. This article is highlighted in the In This Issue feature, p. 517.

In vivo efficacy of platelet-delivered, high specific activity factor VIII variants.

Ectopically expressed, human B-domainless (hB) factor 8 (F8) in platelets improves hemostasis in hemophilia A mice in several injury models. However, in both a cuticular bleeding model and a cremaster laser arteriole/venule injury model, there were limitations to platelet-derived (p) hBF8 efficacy, including increased clot embolization. We now address whether variants of F8 with enhanced activity, inactivation resistant F8 (IR8) and canine (c) BF8, would improve clotting efficacy. In both transgenic and lentiviral murine model approaches, pIR8 expressed at comparable levels to phBF8, but pcBF8 expressed at only approximately 30%. Both variants were more effective than hBF8 in cuticular bleeding and FeCl(3) carotid artery models. However, in the cremaster injury model, only pcBF8 was more effective, markedly decreasing clot embolization. Because inhibitors of F8 are stored in platelet granules and IR8 is not protected by binding to von Willebrand factor, we also tested whether pIR8 was effective in the face of inhibitors and found that pIR8 is protected from the inhibitors. In summary, pF8 variants with high specific activity are more effective in controlling bleeding, but this improved efficacy was inconsistent between bleeding models, perhaps reflecting the underlying mechanism(s) for the increased specific activity of the studied F8 variants.

Ecological restoration stimulates environmental outcomes but exacerbates water shortage in the Loess Plateau.

Restoration is the natural and intervention-assisted set of processes designed to promote and facilitate the recovery of an ecosystem that has been degraded, damaged, or destroyed. However, it can also have an adverse effect on the environment. Thus, assessing an ecological restoration project's impact is crucial to determining its success and optimum management strategies. We performed a meta-analysis concerning the environmental outcomes during the years 2000-2015 resulting from the "Grain for Green" Project (GFGP) implementation in the Loess Plateau (LP). Data were gathered from 40 peer-reviewed English-language articles chosen from a pool of 332 articles. The results showed that, on average, GFGP increased forest coverage by 35.7% (95% CI [24.15-47.52%]), and grassland by 1.05% (95% CI [0.8-1.28%]). At the same time, GFGP has a positive impact on soil carbon (C) sequestration, net ecosystem production (NEP), and net primary production (NPP), from the years 2000 to 2015 by an average of 36% (95% CI [28.96-43.18%]), 22.7% (95% CI [9.10-36.79%]), and 13.5% (95% CI [9.44-17.354%]), respectively. Soil erosion, sediment load, runoff coefficient, and water yield were reduced by 13.3% (95% CI [0.27-25.76%]), 21.5% (95% CI [1.50-39.99%]), 22.4% (95% CI [5.28-40.45%]) and 43.3% (95% CI [27.03-82.86%]), respectively, from the years 2000 to 2015. Our results indicate that water supply decreased with the increase of vegetation coverage. Therefore, to balance the needs for green space, GFGP policies and strategies should recover, enhance, and sustain more resilient ecosystems.

Correction: Ecological restoration stimulates environmental outcomes but exacerbates water shortage in the Loess Plateau.

[This corrects the article DOI: 10.7717/peerj.13658.].

Antioxidants reduce endoplasmic reticulum stress and improve protein secretion.

Protein misfolding in the endoplasmic reticulum (ER) contributes to the pathogenesis of many diseases. Although oxidative stress can disrupt protein folding, how protein misfolding and oxidative stress impact each other has not been explored. We have analyzed expression of coagulation factor VIII (FVIII), the protein deficient in hemophilia A, to elucidate the relationship between protein misfolding and oxidative stress. Newly synthesized FVIII misfolds in the ER lumen, activates the unfolded protein response (UPR), causes oxidative stress, and induces apoptosis in vitro and in vivo in mice. Strikingly, antioxidant treatment reduces UPR activation, oxidative stress, and apoptosis, and increases FVIII secretion in vitro and in vivo. The findings indicate that reactive oxygen species are a signal generated by misfolded protein in the ER that cause UPR activation and cell death. Genetic or chemical intervention to reduce reactive oxygen species improves protein folding and cell survival and may provide an avenue to treat and/or prevent diseases of protein misfolding.

Intensive vegetable production results in high nitrate accumulation in deep soil profiles in China.

A comprehensive understanding of the patterns and controlling factors of nitrate accumulation in intensive vegetable production is essential to solve this problem. For the first time, the national patterns and controlling factors of nitrate accumulation in soil of vegetable systems in China were analysed by compiling 1262 observations from 117 published articles. The results revealed that the nitrate accumulation at 0-100 cm, 100-200 cm, 200-300 cm, and >300 cm were 504, 390, 349, and 244 kg N ha-1, with accumulation rates of 62, 54, 19, and 16 kg N ha-1 yr-1 for plastic greenhouse vegetables (PG); for open field vegetables (OF), they were 264, 217, 228, and 242 kg N ha-1 with accumulation rates of 26, 24, 18, and 10 kg N ha-1 yr-1, respectively. Nitrate accumulation at 0-100 cm, 0-200 cm, and 0-400 cm accounted for 5%, 11%, and 17% of accumulated nitrogen (N) inputs for PG, and represented 4%, 9%, and 13% of accumulated N inputs for OF. Nitrogen input rates and soil pH had positive effects and soil organic carbon, water input rate, and carbon to nitrogen ratio (C/N) had negative effects on nitrate accumulation in root zone (0-100 cm soil). Nitrate accumulation in deep vadose zone (>100 cm soil) was positively correlated with N and water input rates, and was negatively correlated with soil organic carbon, C/N, and the clay content. Thus, for a given vegetable soil with relatively stable soil pH and soil clay content, reducing N and water inputs, and increasing soil organic carbon and C/N are effective measures to control nitrate accumulation.

Discovery of first-in-class inhibitors of ASH1L histone methyltransferase with anti-leukemic activity.

ASH1L histone methyltransferase plays a crucial role in the pathogenesis of different diseases, including acute leukemia. While ASH1L represents an attractive drug target, developing ASH1L inhibitors is challenging, as the catalytic SET domain adapts an inactive conformation with autoinhibitory loop blocking the access to the active site. Here, by applying fragment-based screening followed by medicinal chemistry and a structure-based design, we developed first-in-class small molecule inhibitors of the ASH1L SET domain. The crystal structures of ASH1L-inhibitor complexes reveal compound binding to the autoinhibitory loop region in the SET domain. When tested in MLL leukemia models, our lead compound, AS-99, blocks cell proliferation, induces apoptosis and differentiation, downregulates MLL fusion target genes, and reduces the leukemia burden in vivo. This work validates the ASH1L SET domain as a druggable target and provides a chemical probe to further study the biological functions of ASH1L as well as to develop therapeutic agents.

Menin inhibitor MI-3454 induces remission in MLL1-rearranged and NPM1-mutated models of leukemia.

The protein-protein interaction between menin and mixed lineage leukemia 1 (MLL1) plays a critical role in acute leukemias with translocations of the MLL1 gene or with mutations in the nucleophosmin 1 (NPM1) gene. As a step toward clinical translation of menin-MLL1 inhibitors, we report development of MI-3454, a highly potent and orally bioavailable inhibitor of the menin-MLL1 interaction. MI-3454 profoundly inhibited proliferation and induced differentiation in acute leukemia cells and primary patient samples with MLL1 translocations or NPM1 mutations. When applied as a single agent, MI-3454 induced complete remission or regression of leukemia in mouse models of MLL1-rearranged or NPM1-mutated leukemia, including patient-derived xenograft models, through downregulation of key genes involved in leukemogenesis. We also identified MEIS1 as a potential pharmacodynamic biomarker of treatment response with MI-3454 in leukemia, and demonstrated that this compound is well tolerated and did not impair normal hematopoiesis in mice. Overall, this study demonstrates, for the first time to our knowledge, profound activity of the menin-MLL1 inhibitor as a single agent in clinically relevant PDX models of leukemia. These data provide a strong rationale for clinical translation of MI-3454 or its analogs for leukemia patients with MLL1 rearrangements or NPM1 mutations.

GAS41 Recognizes Diacetylated Histone H3 through a Bivalent Binding Mode.

GAS41 is a chromatin-associated protein that belongs to the YEATS family and is involved in the recognition of acetyl-lysine in histone proteins. A unique feature of GAS41 is the presence of a C-terminal coiled-coil domain, which is responsible for protein dimerization. Here, we characterized the specificity of the GAS41 YEATS domain and found that it preferentially binds to acetylated H3K18 and H3K27 peptides. Interestingly, we found that full-length, dimeric GAS41 binds to diacetylated H3 peptides with an enhanced affinity when compared to those for monoacetylated peptides, through a bivalent binding mode. We determined the crystal structure of the GAS41 YEATS domain with H3K23acK27ac to visualize the molecular basis of diacetylated histone binding. Our results suggest a unique binding mode in which full-length GAS41 is a reader of diacetylated histones.