CRISPR/Cas9-Mediated Foxp1 Silencing Restores Immune Surveillance in an Immunocompetent A20 Lymphoma Model.

The interaction of lymphoma cells with their microenvironment has an important role in disease pathogenesis and is being actively pursued therapeutically using immunomodulatory drugs, including immune checkpoint inhibitors. Diffuse large B-cell lymphoma (DLBCL) is an aggressive high-grade disease that remains incurable in ~40% of patients treated with R-CHOP immunochemotherapy. The FOXP1 transcription factor is abundantly expressed in such high-risk DLBCL and we recently identified its regulation of immune response signatures, in particular, its suppression of the cell surface expression of major histocompatibility class II (MHC-II), which has a critical role in antigen presentation to T cells. Using CRISPR/Cas9 genome editing we have depleted Foxp1 expression in the aggressive murine A20 lymphoma cell line. When grown in BALB/c mice, this cell line provides a high-fidelity immunocompetent disseminated lymphoma model that displays many characteristics of human DLBCL. Transient Foxp1-depletion using siRNA, and stable depletion using CRISPR (generated by independently targeting Foxp1 exon six or seven) upregulated cell surface I-Ab (MHC-II) expression without impairing cell viability in vitro. RNA sequencing of Foxp1-depleted A20 clones identified commonly deregulated genes, such as the B-cell marker Cd19, and hallmark DLBCL signatures such as MYC-targets and oxidative phosphorylation. Immunocompetent animals bearing Foxp1-depleted A20 lymphomas showed significantly-improved survival, and 20% did not develop tumors; consistent with modulating immune surveillance, this was not observed in immunodeficient NOD SCIDγ mice. The A20 Foxp1 CRISPR model will help to further characterize the contribution of Foxp1 to lymphoma immune evasion and the potential for Foxp1 targeting to synergize with other immunotherapies.

Response of high-risk MDS to azacitidine and lenalidomide is impacted by baseline and acquired mutations in a cluster of three inositide-specific genes.

Specific myeloid-related and inositide-specific gene mutations can be linked to myelodysplastic syndromes (MDS) pathogenesis and therapy. Here, 44 higher-risk MDS patients were treated with azacitidine and lenalidomide and mutations analyses were performed at baseline and during the therapy. Results were then correlated to clinical outcome, overall survival (OS), leukemia-free-survival (LFS) and response to therapy. Collectively, 34/44 patients were considered evaluable for response, with an overall response rate of 76.25% (26/34 cases): 17 patients showed a durable response, 9 patients early lost response and 8 patients never responded. The most frequently mutated genes were ASXL1, TET2, RUNX1, and SRSF2. All patients early losing response, as well as cases never responding, acquired the same 3 point mutations during therapy, affecting respectively PIK3CD (D133E), AKT3 (D280G), and PLCG2 (Q548R) genes, that regulate cell proliferation and differentiation. Moreover, Kaplan-Meier analyses revealed that this mutated cluster was significantly associated with a shorter OS, LFS, and duration of response. All in all, a common mutated cluster affecting 3 inositide-specific genes is significantly associated with loss of response to azacitidine and lenalidomide therapy in higher risk MDS. Further studies are warranted to confirm these data and to further analyze the functional role of this 3-gene cluster.

Impact of spliceosome mutations on RNA splicing in myelodysplasia: dysregulated genes/pathways and clinical associations.

SF3B1, SRSF2, and U2AF1 are the most frequently mutated splicing factor genes in the myelodysplastic syndromes (MDS). We have performed a comprehensive and systematic analysis to determine the effect of these commonly mutated splicing factors on pre-mRNA splicing in the bone marrow stem/progenitor cells and in the erythroid and myeloid precursors in splicing factor mutant MDS. Using RNA-seq, we determined the aberrantly spliced genes and dysregulated pathways in CD34+ cells of 84 patients with MDS. Splicing factor mutations result in different alterations in splicing and largely affect different genes, but these converge in common dysregulated pathways and cellular processes, focused on RNA splicing, protein synthesis, and mitochondrial dysfunction, suggesting common mechanisms of action in MDS. Many of these dysregulated pathways and cellular processes can be linked to the known disease pathophysiology associated with splicing factor mutations in MDS, whereas several others have not been previously associated with MDS, such as sirtuin signaling. We identified aberrantly spliced events associated with clinical variables, and isoforms that independently predict survival in MDS and implicate dysregulation of focal adhesion and extracellular exosomes as drivers of poor survival. Aberrantly spliced genes and dysregulated pathways were identified in the MDS-affected lineages in splicing factor mutant MDS. Functional studies demonstrated that knockdown of the mitosis regulators SEPT2 and AKAP8, aberrantly spliced target genes of SF3B1 and SRSF2 mutations, respectively, led to impaired erythroid cell growth and differentiation. This study illuminates the effect of the common spliceosome mutations on the MDS phenotype and provides novel insights into disease pathophysiology.

Splicing factor mutations in the myelodysplastic syndromes: target genes and therapeutic approaches.

Mutations in splicing factor genes (SF3B1, SRSF2, U2AF1 and ZRSR2) are frequently found in patients with myelodysplastic syndromes (MDS), suggesting that aberrant spliceosome function plays a key role in the pathogenesis of MDS. Splicing factor mutations have been shown to result in aberrant splicing of many downstream target genes. Recent functional studies have begun to characterize the splicing dysfunction in MDS, identifying some key aberrantly spliced genes that are implicated in disease pathophysiology. These findings have led to the development of therapeutic strategies using splicing-modulating agents and rapid progress is being made in this field. Splicing inhibitors are promising agents that exploit the preferential sensitivity of splicing factor-mutant cells to these compounds. Here, we review the known target genes associated with splicing factor mutations in MDS, and discuss the potential of splicing-modulating therapies for these disorders.

Structural Dynamics of 15-Lipoxygenase-2 via Hydrogen-Deuterium Exchange.

Eicosanoids are inflammatory signaling lipids that are biosynthesized in response to cellular injury or threat. They were originally thought to be pro-inflammatory molecules, but members of at least one subclass, the lipoxins, are able to resolve inflammation. One step in lipoxin synthesis is the oxygenation of arachidonic acid by 15-lipoxygenase (15-LOX). 15-LOX contains two domains: a Ca2+ binding PLAT domain and a catalytic domain. 15-LOX is a soluble cytosolic protein until binding of Ca2+ to the PLAT domain promotes translocation to the membrane surface. The role of 15-LOX structural dynamics in this translocation has remained unclear. We investigated the dynamics of 15-LOX isoform B (15-LOX-2) upon binding of Ca2+ and ligands, as well as upon membrane association using hydrogen-deuterium exchange mass spectrometry (HDX-MS). We used HDX-MS to probe the solvent accessibility and backbone flexibility of 15-LOX-2, revealing significant differences in deuterium incorporation between the PLAT and catalytic domains, with the PLAT domain demonstrating higher flexibility. Comparison of HDX for 15-LOX-2 in the presence and absence of Ca2+ indicates there are few differences in structural dynamics. Furthermore, our HDX results involving nanodisc-associated 15-LOX-2 suggest that significant structural and dynamic changes in 15-LOX-2 are not required for membrane association. Our results also show that a substrate lipid binding to the active site in the catalytic domain does induce changes in incorporation of deuterium into the PLAT domain. Overall, our results challenge the previous hypothesis that Ca2+ binding induces major structural changes in the PLAT domain and support the hypothesis that is interdomain communication in 15-LOX-2.

Global Kinetic Mechanism of Microsomal Glutathione Transferase 1 and Insights into Dynamic Enzyme Activation.

Microsomal glutathione transferase 1 (MGST1) has a unique ability to be activated, ≤30-fold, by modification with sulfhydryl reagents. MGST1 exhibits one-third-of-the-sites reactivity toward glutathione and hence heterogeneous binding to different active sites in the homotrimer. Limited turnover stopped-flow kinetic measurements of the activated enzyme allowed us to more accurately determine the KD for the "third" low-affinity GSH binding site (1.4 ± 0.3 mM). The rate of thiolate formation, k2 (0.77 ± 0.06 s-1), relevant to turnover, could also be determined. By deriving the steady-state rate equation for a random sequential mechanism for MGST1, we can predict KM, kcat, and kcat/KM values from these and previously determined pre-steady-state rate constants (all determined at 5 °C). To assess whether the pre-steady-state behavior can account for the steady-state kinetic behavior, we have determined experimental values for kinetic parameters at 5 °C. For reactive substrates and the activated enzyme, data for the microscopic steps account for the global mechanism of MGST1. For the unactivated enzyme and more reactive electrophilic substrates, pre-steady-state and steady-state data can be reconciled only if a more active subpopulation of MGST1 is assumed. We suggest that unactivated MGST1 can be partially activated in its unmodified form. The existence of an activated subpopulation (approximately 10%) could be demonstrated in limited turnover experiments. We therefore suggest that MSGT1 displays a preexisting dynamic equilibrium between high- and low-activity forms.

The U2AF1S34F mutation induces lineage-specific splicing alterations in myelodysplastic syndromes.

Mutations of the splicing factor-encoding gene U2AF1 are frequent in the myelodysplastic syndromes (MDS), a myeloid malignancy, and other cancers. Patients with MDS suffer from peripheral blood cytopenias, including anemia, and an increasing percentage of bone marrow myeloblasts. We studied the impact of the common U2AF1S34F mutation on cellular function and mRNA splicing in the main cell lineages affected in MDS. We demonstrated that U2AF1S34F expression in human hematopoietic progenitors impairs erythroid differentiation and skews granulomonocytic differentiation toward granulocytes. RNA sequencing of erythroid and granulomonocytic colonies revealed that U2AF1S34F induced a higher number of cassette exon splicing events in granulomonocytic cells than in erythroid cells. U2AF1S34F altered mRNA splicing of many transcripts that were expressed in both cell types in a lineage-specific manner. In hematopoietic progenitors, the introduction of isoform changes identified in the U2AF1S34F target genes H2AFY, encoding an H2A histone variant, and STRAP, encoding serine/threonine kinase receptor-associated protein, recapitulated phenotypes associated with U2AF1S34F expression in erythroid and granulomonocytic cells, suggesting a causal link. Furthermore, we showed that isoform modulation of H2AFY and STRAP rescues the erythroid differentiation defect in U2AF1S34F MDS cells, suggesting that splicing modulators could be used therapeutically. These data have critical implications for understanding MDS phenotypic heterogeneity and support the development of therapies targeting splicing abnormalities.

Arg126 and Asp49 Are Essential for the Catalytic Function of Microsomal Prostaglandin E2 Synthase 1 and Ser127 Is Not.

INTRODUCTION: Prostaglandins are signaling molecules that regulate different physiological processes, involving allergic and inflammatory responses and cardiovascular control. They are involved in several pathophysiological processes, including inflammation and cancer. The inducible terminal enzyme, microsomal prostaglandin E synthase 1 (MPGES1), catalyses prostaglandin E2 production during inflammation. MPGES1 has therefore been intensively studied as a pharmaceutical target and many competitive inhibitors targeting its active site have been developed. However, little is known about its catalytic mechanism. AIM: The objective of this study was to investigate which amino acids play a key role in the catalytic mechanism of MPGES1. MATERIALS AND METHODS: Based on results and predictions from previous structural studies, the amino acid residues Asp49, Arg73, Arg126, and Ser127 were chosen and altered by site-directed mutagenesis. The mutated enzyme variants were cloned and expressed in both the E. coli and the Baculovirus expression systems. Their catalytic significance was evaluated by activity measurements with prostanoid profiling. RESULTS AND CONCLUSIONS: Our study shows that Arg126 and Asp49 are absolutely required for the catalytic activity of MPGES1, as when exchanged, the enzyme variants loose activity. Ser127 and Arg73 on the other hand, don't seem to be central to the catalytic mechanism because when exchanged, their variants retain considerable activity. Our finding that the Ser127Ala variant retains activity was surprising since high-resolution structural data supported a role in glutathione activation. The close proximity of Ser127 to the active site is, however, supported since the Ser127Cys variant displays 80% lowered activity.

Staphylococcus aureus CstB Is a Novel Multidomain Persulfide Dioxygenase-Sulfurtransferase Involved in Hydrogen Sulfide Detoxification.

Hydrogen sulfide (H2S) is both a lethal gas and an emerging gasotransmitter in humans, suggesting that the cellular H2S level must be tightly regulated. CstB is encoded by the cst operon of the major human pathogen Staphylococcus aureus and is under the transcriptional control of the persulfide sensor CstR and H2S. Here, we show that CstB is a multifunctional Fe(II)-containing persulfide dioxygenase (PDO), analogous to the vertebrate protein ETHE1 (ethylmalonic encephalopathy protein 1). Chromosomal deletion of ethe1 is fatal in vertebrates. In the presence of molecular oxygen (O2), hETHE1 oxidizes glutathione persulfide (GSSH) to generate sulfite and reduced glutathione. In contrast, CstB oxidizes major cellular low molecular weight (LMW) persulfide substrates from S. aureus, coenzyme A persulfide (CoASSH) and bacillithiol persulfide (BSSH), directly to generate thiosulfate (TS) and reduced thiols, thereby avoiding the cellular toxicity of sulfite. Both Cys201 in the N-terminal PDO domain (CstB(PDO)) and Cys408 in the C-terminal rhodanese domain (CstB(Rhod)) strongly enhance the TS generating activity of CstB. CstB also possesses persulfide transferase (PT; reverse rhodanese) activity, which generates TS when provided with LMW persulfides and sulfite, as well as conventional thiosulfate transferase (TST; rhodanese) activity; both of these activities require Cys408. CstB protects S. aureus against H2S toxicity, with the C201S and C408S cstB genes being unable to rescue a NaHS-induced ΔcstB growth phenotype. Induction of the cst operon by NaHS reveals that functional CstB impacts cellular TS concentrations. These data collectively suggest that CstB may have evolved to facilitate the clearance of LMW persulfides that occur upon elevation of the level of cellular H2S and hence may have an impact on bacterial viability under H2S misregulation, in concert with the other enzymes encoded by the cst operon.

Prediction of substrates for glutathione transferases by covalent docking.

Enzymes in the glutathione transferase (GST) superfamily catalyze the conjugation of glutathione (GSH) to electrophilic substrates. As a consequence they are involved in a number of key biological processes, including protection of cells against chemical damage, steroid and prostaglandin biosynthesis, tyrosine catabolism, and cell apoptosis. Although virtual screening has been used widely to discover substrates by docking potential noncovalent ligands into active site clefts of enzymes, docking has been rarely constrained by a covalent bond between the enzyme and ligand. In this study, we investigate the accuracy of docking poses and substrate discovery in the GST superfamily, by docking 6738 potential ligands from the KEGG and MetaCyc compound libraries into 14 representative GST enzymes with known structures and substrates using the PLOP program [ Jacobson Proteins 2004 , 55 , 351 ]. For X-ray structures as receptors, one of the top 3 ranked models is within 3 Å all-atom root mean square deviation (RMSD) of the native complex in 11 of the 14 cases; the enrichment LogAUC value is better than random in all cases, and better than 25 in 7 of 11 cases. For comparative models as receptors, near-native ligand-enzyme configurations are often sampled but difficult to rank highly. For models based on templates with the highest sequence identity, the enrichment LogAUC is better than 25 in 5 of 11 cases, not significantly different from the crystal structures. In conclusion, we show that covalent docking can be a useful tool for substrate discovery and point out specific challenges for future method improvement.

Large-scale determination of sequence, structure, and function relationships in cytosolic glutathione transferases across the biosphere.

The cytosolic glutathione transferase (cytGST) superfamily comprises more than 13,000 nonredundant sequences found throughout the biosphere. Their key roles in metabolism and defense against oxidative damage have led to thousands of studies over several decades. Despite this attention, little is known about the physiological reactions they catalyze and most of the substrates used to assay cytGSTs are synthetic compounds. A deeper understanding of relationships across the superfamily could provide new clues about their functions. To establish a foundation for expanded classification of cytGSTs, we generated similarity-based subgroupings for the entire superfamily. Using the resulting sequence similarity networks, we chose targets that broadly covered unknown functions and report here experimental results confirming GST-like activity for 82 of them, along with 37 new 3D structures determined for 27 targets. These new data, along with experimentally known GST reactions and structures reported in the literature, were painted onto the networks to generate a global view of their sequence-structure-function relationships. The results show how proteins of both known and unknown function relate to each other across the entire superfamily and reveal that the great majority of cytGSTs have not been experimentally characterized or annotated by canonical class. A mapping of taxonomic classes across the superfamily indicates that many taxa are represented in each subgroup and highlights challenges for classification of superfamily sequences into functionally relevant classes. Experimental determination of disulfide bond reductase activity in many diverse subgroups illustrate a theme common for many reaction types. Finally, sequence comparison between an enzyme that catalyzes a reductive dechlorination reaction relevant to bioremediation efforts with some of its closest homologs reveals differences among them likely to be associated with evolution of this unusual reaction. Interactive versions of the networks, associated with functional and other types of information, can be downloaded from the Structure-Function Linkage Database (SFLD; http://sfld.rbvi.ucsf.edu).

Structure and function of the genomically encoded fosfomycin resistance enzyme, FosB, from Staphylococcus aureus.

The Gram-positive pathogen Staphylococcus aureus is a leading cause of global morbidity and mortality. Like many multi-drug-resistant organisms, S. aureus contains antibiotic-modifying enzymes that facilitate resistance to a multitude of antimicrobial compounds. FosB is a Mn(2+)-dependent fosfomycin-inactivating enzyme found in S. aureus that catalyzes nucleophilic addition of either l-cysteine (l-Cys) or bacillithiol (BSH) to the antibiotic, resulting in a modified compound with no bactericidal properties. The three-dimensional X-ray crystal structure of FosB from S. aureus (FosB(Sa)) has been determined to a resolution of 1.15 Å. Cocrystallization of FosB(Sa) with either l-Cys or BSH results in a disulfide bond between the exogenous thiol and the active site Cys9 of the enzyme. An analysis of the structures suggests that a highly conserved loop region of the FosB enzymes must change conformation to bind fosfomycin. While two crystals of FosB(Sa) contain Zn(2+) in the active site, kinetic analyses of FosB(Sa) indicated that the enzyme is inhibited by Zn(2+) for l-Cys transferase activity and only marginally active for BSH transferase activity. Fosfomycin-treated disk diffusion assays involving S. aureus Newman and the USA300 JE2 methicillin-resistant S. aureus demonstrate a marked increase in the sensitivity of the organism to the antibiotic in either the BSH or FosB null strains, indicating that both are required for survival of the organism in the presence of the antibiotic. This work identifies FosB as a primary fosfomycin-modifying pathway of S. aureus and establishes the enzyme as a potential therapeutic target for increased efficacy of fosfomycin against the pathogen.

Structural and chemical aspects of resistance to the antibiotic fosfomycin conferred by FosB from Bacillus cereus.

The fosfomycin resistance enzymes, FosB, from Gram-positive organisms, are M(2+)-dependent thiol tranferases that catalyze nucleophilic addition of either L-cysteine (L-Cys) or bacillithiol (BSH) to the antibiotic, resulting in a modified compound with no bacteriacidal properties. Here we report the structural and functional characterization of FosB from Bacillus cereus (FosB(Bc)). The overall structure of FosB(Bc), at 1.27 Å resolution, reveals that the enzyme belongs to the vicinal oxygen chelate (VOC) superfamily. Crystal structures of FosB(Bc) cocrystallized with fosfomycin and a variety of divalent metals, including Ni(2+), Mn(2+), Co(2+), and Zn(2+), indicate that the antibiotic coordinates to the active site metal center in an orientation similar to that found in the structurally homologous manganese-dependent fosfomycin resistance enzyme, FosA. Surface analysis of the FosB(Bc) structures show a well-defined binding pocket and an access channel to C1 of fosfomycin, the carbon to which nucleophilic addition of the thiol occurs. The pocket and access channel are appropriate in size and shape to accommodate L-Cys or BSH. Further investigation of the structures revealed that the fosfomycin molecule, anchored by the metal, is surrounded by a cage of amino acids that hold the antibiotic in an orientation such that C1 is centered at the end of the solvent channel, positioning the compound for direct nucleophilic attack by the thiol substrate. In addition, the structures of FosB(Bc) in complex with the L-Cys-fosfomycin product (1.55 Å resolution) and in complex with the bacillithiol-fosfomycin product (1.77 Å resolution) coordinated to a Mn(2+) metal in the active site have been determined. The L-Cys moiety of either product is located in the solvent channel, where the thiol has added to the backside of fosfomycin C1 located at the end of the channel. Concomitant kinetic analyses of FosB(Bc) indicated that the enzyme has a preference for BSH over L-Cys when activated by Mn(2+) and is inhibited by Zn(2+). The fact that Zn(2+) is an inhibitor of FosB(Bc) was used to obtain a ternary complex structure of the enzyme with both fosfomycin and L-Cys bound.

Synthesis of bacillithiol and the catalytic selectivity of FosB-type fosfomycin resistance proteins.

Bacillithiol (BSH) has been prepared on the gram scale from the inexpensive starting material, D-glucosamine hydrochloride, in 11 steps and 8-9% overall yield. The BSH was used to survey the substrate and metal-ion selectivity of FosB enzymes from four Gram-positive microorganisms associated with the deactivation of the antibiotic fosfomycin. The in vitro results indicate that the preferred thiol substrate and metal ion for the FosB from Staphylococcus aureus are BSH and Ni(II), respectively. However, the metal-ion selectivity is less distinct with FosB from Bacillus subtilis, Bacillus anthracis, or Bacillus cereus.

Differential TERT promoter methylation and response to 5-aza-2'-deoxycytidine in acute myeloid leukemia cell lines: TERT expression, telomerase activity, telomere length, and cell death.

The catalytic subunit of human telomerase (TERT) is highly expressed in cancer cells, and correlates with complex cytogenetics and disease severity in acute myeloid leukemia (AML). The TERT promoter is situated within a large CpG island, suggesting that expression is methylation-sensitive. Studies suggest a correlation between hypermethylation and TERT overexpression. We investigated the relationship between TERT promoter methylation and expression and telomerase activity in human leukemia and lymphoma cell lines. DAC-induced demethylation and cell death were observed in all three cell lines, as well as telomere shortening in HL-60 cells. DAC treatment reduced TERT expression and telomerase activity in OCI/AML3 and HL-60 cells, but not in U937 cells. Control U937 cells expressed lower levels of TERT mRNA, carried a highly methylated TERT core promoter, and proved more resistant to DAC-induced repression of TERT expression and cell death. AML patients had significantly lower methylation levels at several CpGs than "well elderly" individuals. This study, the first to investigate the relationship between TERT methylation and telomerase activity in leukemia cells, demonstrated a differential methylation pattern and response to DAC in three AML cell lines. We suggest that, although DAC treatment reduces TERT expression and telomerase activity, this is unlikely to occur via direct demethylation of the TERT promoter. However, further investigations on the regions spanning CpGs 7-12 and 14-16 may reveal valuable information regarding transcriptional regulation of TERT.