Laurens Kruidenier

A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response

The jumonji (JMJ) family of histone demethylases are Fe2+- and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes. These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context. Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance, as well as in development, physiology and disease. However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX). The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family.

Contrasting roles of histone 3 lysine 27 demethylases in acute lymphoblastic leukaemia.

T-cell acute lymphoblastic leukaemia (T-ALL) is a haematological malignancy with a dismal overall prognosis, including a relapse rate of up to 25%, mainly because of the lack of non-cytotoxic targeted therapy options. Drugs that target the function of key epigenetic factors have been approved in the context of haematopoietic disorders, and mutations that affect chromatin modulators in a variety of leukaemias have recently been identified; however, ‘epigenetic’ drugs are not currently used for T-ALL treatment. Recently, we described that the polycomb repressive complex 2 (PRC2) has a tumour-suppressor role in T-ALL. Here we delineated the role of the histone 3 lysine 27 (H3K27) demethylases JMJD3 and UTX in T-ALL. We show that JMJD3 is essential for the initiation and maintenance of T-ALL, as it controls important oncogenic gene targets by modulating H3K27 methylation. By contrast, we found that UTX functions as a tumour suppressor and is frequently genetically inactivated in T-ALL. Moreover, we demonstrated that the small molecule inhibitor GSKJ4 (ref. 5) affects T-ALL growth, by targeting JMJD3 activity. These findings show that two proteins with a similar enzymatic function can have opposing roles in the context of the same disease, paving the way for treating haematopoietic malignancies with a new category of epigenetic inhibitors.

Catalytic in vivo protein knockdown by small-molecule PROTACs

The current predominant therapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the targets ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR.

Selective class IIa histone deacetylase inhibition via a nonchelating zinc-binding group

In contrast to studies on class I histone deacetylase (HDAC) inhibitors, the elucidation of the molecular mechanisms and therapeutic potential of class IIa HDACs (HDAC4, HDAC5, HDAC7 and HDAC9) is impaired by the lack of potent and selective chemical probes. Here we report the discovery of inhibitors that fill this void with an unprecedented metal-binding group, trifluoromethyloxadiazole (TFMO), which circumvents the selectivity and pharmacologic liabilities of hydroxamates. We confirm direct metal binding of the TFMO through crystallographic approaches and use chemoproteomics to demonstrate the superior selectivity of the TFMO series relative to a hydroxamate-substituted analog. We further apply these tool compounds to reveal gene regulation dependent on the catalytic active site of class IIa HDACs. The discovery of these inhibitors challenges the design process for targeting metalloenzymes through a chelating metal-binding group and suggests therapeutic potential for class IIa HDAC enzyme blockers distinct in mechanism and application compared to current HDAC inhibitors.

Targeting Gut T Cell Ca2+ Release-Activated Ca2+ Channels Inhibits T Cell Cytokine Production and T-Box Transcription Factor T-Bet in Inflammatory Bowel Disease

Prolonged Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels is crucial in activating the Ca2+-sensitive transcription factor NFAT, which is responsible for directing T cell proliferation and cytokine gene expression. To establish whether targeting CRAC might counteract intestinal inflammation, we evaluated the in vitro effect of a selective CRAC inhibitor on T cell cytokine production and T-bet expression by lamina propria mononuclear cells (LPMC) and biopsy specimens from inflammatory bowel disease (IBD) patients. The inhibitory activity of the CRAC blocker was investigated through patch-clamp experiments on rat basophilic leukemia cells and fluorometric imaging plate reader intracellular Ca2+ assays using thapsigargin-stimulated Jurkat T cells and its detailed selectivity profile defined using a range of in vitro radioligand binding and functional assays. Anti-CD3/CD28-stimulated LPMC and biopsy specimens from 51 patients with IBD were cultured with a range of CRAC inhibitor concentrations (0.01–10 μM). IFN-γ, IL-2, IL-8, and IL-17 were analyzed by ELISA. T-bet was determined by immunoblotting. We found that the CRAC blocker concentration-dependently inhibited CRAC current in rat basophilic leukemia cells and thapsigargin-induced Ca2+ influx in Jurkat T cells. A concentration-dependent reduction in T-bet expression and production of IFN-γ, IL-2, IL-17, but not IL-8, was observed in IBD LPMC and biopsy specimens treated with the CRAC inhibitor. In conclusion, we provide evidence that the suppression of CRAC channel function may dampen the increased T cell response in the inflamed gut, thus suggesting a promising role for CRAC inhibitor drugs in the therapeutic management of patients with IBD.

Blockade of transforming growth factor β upregulates T-box transcription factor T-bet, and increases T helper cell type 1 cytokine and matrix metalloproteinase-3 production in the human gut mucosa

Background and Aims: The role of transforming growth factor β (TGFβ) in inhibiting T cell function in the normal gut has been studied in animal models. However, the impact of TGFβ inhibition on T cells in the normal human gut remains poorly understood. The effect of TGFβ blockade in normal intestinal biopsies grown ex vivo and lamina propria mononuclear cells (LPMCs) on T-bet, a T-box transcription factor required for T helper cell type (Th)1 differentiation, interferon γ (IFNγ) production, T cell apoptosis and matrix metalloproteinase (MMP)-3 production has therefore been tested. Methods: TGFβ transcripts were determined by quantitative reverse transcription-PCR in laser-captured gut epithelium and lamina propria. Biopsies and LPMCs were cultured with anti-TGFβ neutralising antibody. After 24 h culture, T-bet was determined by immunoblotting, and T cell apoptosis was assessed by flow cytometry. IFNγ, tumour necrosis factor α (TNFα), interleukin (IL) 2, IL6, IL8, IL10, IL12p70 and IL17 were measured by ELISA. MMP-3 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 were assessed by immunoblotting. Results: A higher number of TGFβ transcripts was found in the lamina propria than in the epithelium in normal gut. T-bet expression was significantly higher in biopsies and LPMCs cultured with anti-TGFβ antibody than in those cultured with control antibody. TGFβ blockade downregulated T cell apoptosis, and induced a significant increase in IFNγ, TNFα, IL2, IL6, IL8 and IL17 production. A higher expression of MMP-3, but not TIMP-1, was observed in the tissue and supernatant of biopsies treated with anti-TGFβ antibody. Conclusions: The findings support a crucial role for TGFβ in dampening T cell-mediated tissue-damaging responses in the human gut.

The H3K27 Demethylase JMJD3 Is Required for Maintenance of the Embryonic Respiratory Neuronal Network, Neonatal Breathing, and Survival

JMJD3 (KDM6B) antagonizes Polycomb silencing by demethylating lysine 27 on histone H3. The interplay of methyltransferases and demethylases at this residue is thought to underlie critical cell fate transitions, and the dynamics of H3K27me3 during neurogenesis posited for JMJD3 a critical role in the acquisition of neural fate. Despite evidence of its involvement in early neural commitment, however, its role in the emergence and maturation of the mammalian CNS remains unknown. Here, we inactivated Jmjd3 in the mouse and found that its loss causes perinatal lethality with the complete and selective disruption of the pre-Bötzinger complex (PBC), the pacemaker of the respiratory rhythm generator. Through genetic and electrophysiological approaches, we show that the enzymatic activity of JMJD3 is selectively required for the maintenance of the PBC and controls critical regulators of PBC activity, uncovering an unanticipated role of this enzyme in the late structuring and function of neuronal networks.

Role of NleH, a type III secreted effector from attaching and effacing pathogens, in colonization of the bovine, ovine, and murine gut

ABSTRACT The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 colonizes human and animal gut via formation of attaching and effacing lesions. EHEC strains use a type III secretion system to translocate a battery of effector proteins into the mammalian host cell, which subvert diverse signal transduction pathways implicated in actin dynamics, phagocytosis, and innate immunity. The genomes of sequenced EHEC O157:H7 strains contain two copies of the effector protein gene nleH, which share 49% sequence similarity with the gene for the Shigella effector OspG, recently implicated in inhibition of migration of the transcriptional regulator NF-κB to the nucleus. In this study we investigated the role of NleH during EHEC O157:H7 infection of calves and lambs. We found that while EHEC ΔnleH colonized the bovine gut more efficiently than the wild-type strain, in lambs the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. Using the mouse pathogen Citrobacter rodentium, which shares many virulence factors with EHEC O157:H7, including NleH, we observed that the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. We found no measurable differences in T-cell infiltration or hyperplasia in colons of mice inoculated with the wild-type or the nleH mutant strain. Using NF-κB reporter mice carrying a transgene containing a luciferase reporter driven by three NF-κB response elements, we found that NleH causes an increase in NF-κB activity in the colonic mucosa. Consistent with this, we found that the nleH mutant triggered a significantly lower tumor necrosis factor alpha response than the wild-type strain.

Down‐regulation of p38 mitogen‐activated protein kinase activation and proinflammatory cytokine production by mitogen‐activated protein kinase inhibitors in inflammatory bowel disease

Crohns disease and ulcerative colitis are inflammatory bowel diseases (IBD) characterized by chronic relapsing mucosal inflammation. Tumour necrosis factor (TNF)‐α, a known agonist of the mitogen‐activated protein kinase (MAPK) pathway, is a key cytokine in this process. We aimed first to determine whether p38 MAPK is activated in IBD inflamed mucosa, and then studied the effect of four different p38α inhibitory compounds on MAPK phosphorylation and secretion of proinflammatory cytokines by IBD lamina propria mononuclear cells (LPMCs) and organ culture biopsies. In vivo phospho‐p38α and p38α expression was evaluated by immunoblotting on intestinal biopsies from inflamed areas of patients affected by Crohns disease and ulcerative colitis, and from normal mucosa of sex‐ and age‐matched control subjects. Both mucosal biopsies and isolated LPMCs were incubated with four different p38α selective inhibitory drugs. TNF‐α, interleukin (IL)‐1β and IL‐6 were measured in the organ and cell culture supernatants by enzyme‐linked immunosorbent assay. We found higher levels of phospho‐p38α in the inflamed mucosa of IBD patients in comparison to controls. All the p38α inhibitory drugs inhibited p38α phosphorylation and secretion of TNF‐α, IL‐1β and IL‐6 from IBD LPMCs and biopsies. Activated p38α MAPK is up‐regulated in the inflamed mucosa of patients with IBD. Additionally, all the p38α selective inhibitory drugs significantly down‐regulated the activation of the MAPK pathway and the secretion of proinflammatory cytokines.

The p50 Subunit of NF-κB Is Critical for In Vivo Clearance of the Noninvasive Enteric Pathogen Citrobacter rodentium

ABSTRACT Citrobacter rodentium, a natural mouse pathogen, belongs to the family of extracellular enteric pathogens that includes enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC). C. rodentium shares many virulence factors with EPEC and EHEC and relies on attaching-and-effacing lesion formation for colonization and infection of the gut. In vivo, C. rodentium infection is characterized by increased epithelial cell proliferation, mucosal thickening, and a TH1-type immune response, but with protective immunity believed to be mediated by serum immunoglobulin G (IgG). In this work, we characterize the immune response and pathology of mice lacking the p50 subunit of the transcription factor nuclear factor kappa B (NF-κB) during C. rodentium infection. We show that p50−/− mice are unable to clear C. rodentium infection. Furthermore, these animals show a reduced influx of immune cells into infected colonic tissue and greater levels of mucosal hyperplasia and the cytokines tumor necrosis factor alpha and gamma interferon. Surprisingly, despite being unable to eliminate infection, p50−/− mice showed markedly higher levels of anti-Citrobacter IgG and IgM, suggesting that antibody alone is not responsible for bacterial clearance. These data also demonstrate that non-NF-κB-dependent defenses are insufficient to control C. rodentium infection, and hence, the NF-κB p50 subunit is critical for defense against this noninvasive pathogen.