TNRC18 engages H3K9me3 to mediate silencing of endogenous retrotransposons.

Trimethylation of histone H3 lysine 9 (H3K9me3) is crucial for the regulation of gene repression and heterochromatin formation, cell-fate determination and organismal development1. H3K9me3 also provides an essential mechanism for silencing transposable elements1-4. However, previous studies have shown that canonical H3K9me3 readers (for example, HP1 (refs. 5-9) and MPP8 (refs. 10-12)) have limited roles in silencing endogenous retroviruses (ERVs), one of the main transposable element classes in the mammalian genome13. Here we report that trinucleotide-repeat-containing 18 (TNRC18), a poorly understood chromatin regulator, recognizes H3K9me3 to mediate the silencing of ERV class I (ERV1) elements such as LTR12 (ref. 14). Biochemical, biophysical and structural studies identified the carboxy-terminal bromo-adjacent homology (BAH) domain of TNRC18 (TNRC18(BAH)) as an H3K9me3-specific reader. Moreover, the amino-terminal segment of TNRC18 is a platform for the direct recruitment of co-repressors such as HDAC-Sin3-NCoR complexes, thus enforcing optimal repression of the H3K9me3-demarcated ERVs. Point mutagenesis that disrupts the TNRC18(BAH)-mediated H3K9me3 engagement caused neonatal death in mice and, in multiple mammalian cell models, led to derepressed expression of ERVs, which affected the landscape of cis-regulatory elements and, therefore, gene-expression programmes. Collectively, we describe a new H3K9me3-sensing and regulatory pathway that operates to epigenetically silence evolutionarily young ERVs and exert substantial effects on host genome integrity, transcriptomic regulation, immunity and development.

Phase separation drives aberrant chromatin looping and cancer development.

The development of cancer is intimately associated with genetic abnormalities that target proteins with intrinsically disordered regions (IDRs). In human haematological malignancies, recurrent chromosomal translocation of nucleoporin (NUP98 or NUP214) generates an aberrant chimera that invariably retains the nucleoporin IDR-tandemly dispersed repeats of phenylalanine and glycine residues1,2. However, how unstructured IDRs contribute to oncogenesis remains unclear. Here we show that IDRs contained within NUP98-HOXA9, a homeodomain-containing transcription factor chimera recurrently detected in leukaemias1,2, are essential for establishing liquid-liquid phase separation (LLPS) puncta of chimera and for inducing leukaemic transformation. Notably, LLPS of NUP98-HOXA9 not only promotes chromatin occupancy of chimera transcription factors, but also is required for the formation of a broad 'super-enhancer'-like binding pattern typically seen at leukaemogenic genes, which potentiates transcriptional activation. An artificial HOX chimera, created by replacing the phenylalanine and glycine repeats of NUP98 with an unrelated LLPS-forming IDR of the FUS protein3,4, had similar enhancing effects on the genome-wide binding and target gene activation of the chimera. Deeply sequenced Hi-C revealed that phase-separated NUP98-HOXA9 induces CTCF-independent chromatin loops that are enriched at proto-oncogenes. Together, this report describes a proof-of-principle example in which cancer acquires mutation to establish oncogenic transcription factor condensates via phase separation, which simultaneously enhances their genomic targeting and induces organization of aberrant three-dimensional chromatin structure during tumourous transformation. As LLPS-competent molecules are frequently implicated in diseases1,2,4-7, this mechanism can potentially be generalized to many malignant and pathological settings.

Discovery of seven novel putative antigens in membranous nephropathy and membranous lupus nephritis identified by mass spectrometry.

Multiple autoantigens have been identified in membranous nephropathy (MN) by tissue-based proteomics. However, antigenic targets of disease are unknown for over 10% of patients with MN and over half of those with membranous lupus nephritis (MLN). Here, we identified multiple new targets in PLA2R-/THSD7A-/EXT-/NELL1-quadruple negative MN biopsies through mass spectrometry of immune complexes recovered from biopsy tissue of patients with MN. Patients with MN negative for these four antigens were identified from Arkana Laboratories case archives. Protein G immunoprecipitation recovered immune complexes from frozen biopsy tissue from 142 quadruple-negative cases and 278 cases of known antigen type, followed by interrogation by mass spectrometry. Potential putative antigens were confirmed through paraffin immunofluorescence and co-localization with IgG within immune deposits. Consecutive series of 165 cases of PLA2R-negative MN and 142 MLN biopsies were screened to determine the frequency for each potential antigen. Seven putative antigens were discovered within immune complexes from biopsies of patients with MN including FCN3, CD206, EEA1, SEZ6L2, NPR3, MST1, and VASN. Peptides from these proteins were not enriched in the 278 cases of known antigen type. Between three to 30 unique peptides were detected for each new target. Frequencies of each biomarker, determined by staining consecutive case series, ranged from under 1 to 4.9%. NPR3 and CD206 were only positive in index cases. All cases showed co-localization of IgG within the immune deposits. Thus, seven putative antigens were newly identified in MN and MLN. Due to the number of antigens identified, it is becoming impractical to type PLA2R-negative MN or MLN cases through immunostaining alone. A multiplex approach is needed for subtyping of these diseases.

Proprotein convertase subtilisin/kexin type 6 (PCSK6) is a likely antigenic target in membranous nephropathy and nonsteroidal anti-inflammatory drug use.

Drugs are an important secondary cause of membranous nephropathy (MN) with the most common drugs associated with MN being nonsteroidal anti-inflammatory drugs (NSAIDs). Since the target antigen in NSAID-associated MN is not known, we performed laser microdissection of glomeruli followed by mass spectrometry (MS/MS) in 250 cases of PLA2R-negative MN to identify novel antigenic targets. This was followed by immunohistochemistry to localize the target antigen along the glomerular basement membrane and western blot analyses of eluates of frozen biopsy tissue to detect binding of IgG to the novel antigenic target. MS/MS studies revealed high total spectral counts of a novel protein Proprotein Convertase Subtilisin/Kexin Type 6 (PCSK 6) in five of the 250 cases in the discovery cohort. A validation cohort using protein G immunoprecipitation, MS/MS, and immunofluorescence detected PCSK6 in eight additional cases. All cases were negative for known antigens. Ten of 13 cases had a history of heavy NSAID use with no history available in one case. The mean serum creatinine and proteinuria at kidney biopsy were 0.93 ± 0.47 mg/dL and 6.5 ± 3.3 gms/day, respectively. Immunohistochemistry/immunofluorescence showed granular staining for PCSK6 along the glomerular basement membrane, and confocal microscopy showed co-localization of IgG and PCSK6. IgG subclass analysis in three cases revealed codominance of IgG1 and IgG4. Western blot analysis using eluates from frozen tissue showed IgG binding to PCSK6 in PCSK6-associated but not in PLA2R-positive MN. Thus, PCSK6 may be a likely novel antigenic target in MN in patients with prolonged NSAID use.

A conserved BAH module within mammalian BAHD1 connects H3K27me3 to Polycomb gene silencing.

Trimethylation of histone H3 lysine 27 (H3K27me3) is important for gene silencing and imprinting, (epi)genome organization and organismal development. In a prevalent model, the functional readout of H3K27me3 in mammalian cells is achieved through the H3K27me3-recognizing chromodomain harbored within the chromobox (CBX) component of canonical Polycomb repressive complex 1 (cPRC1), which induces chromatin compaction and gene repression. Here, we report that binding of H3K27me3 by a Bromo Adjacent Homology (BAH) domain harbored within BAH domain-containing protein 1 (BAHD1) is required for overall BAHD1 targeting to chromatin and for optimal repression of the H3K27me3-demarcated genes in mammalian cells. Disruption of direct interaction between BAHD1BAH and H3K27me3 by point mutagenesis leads to chromatin remodeling, notably, increased histone acetylation, at its Polycomb gene targets. Mice carrying an H3K27me3-interaction-defective mutation of Bahd1BAH causes marked embryonic lethality, showing a requirement of this pathway for normal development. Altogether, this work demonstrates an H3K27me3-initiated signaling cascade that operates through a conserved BAH 'reader' module within BAHD1 in mammals.

Cistrome analysis of YY1 uncovers a regulatory axis of YY1:BRD2/4-PFKP during tumorigenesis of advanced prostate cancer.

Castration-resistant prostate cancer (CRPC) is a terminal disease and the molecular underpinnings of CRPC development need to be better understood in order to improve its treatment. Here, we report that a transcription factor Yin Yang 1 (YY1) is significantly overexpressed during prostate cancer progression. Functional and cistrome studies of YY1 uncover its roles in promoting prostate oncogenesis in vitro and in vivo, as well as sustaining tumor metabolism including the Warburg effect and mitochondria respiration. Additionally, our integrated genomics and interactome profiling in prostate tumor show that YY1 and bromodomain-containing proteins (BRD2/4) co-occupy a majority of gene-regulatory elements, coactivating downstream targets. Via gene loss-of-function and rescue studies and mutagenesis of YY1-bound cis-elements, we unveil an oncogenic pathway in which YY1 directly binds and activates PFKP, a gene encoding the rate-limiting enzyme for glycolysis, significantly contributing to the YY1-enforced Warburg effect and malignant growth. Altogether, this study supports a master regulator role for YY1 in prostate tumorigenesis and reveals a YY1:BRD2/4-PFKP axis operating in advanced prostate cancer with implications for therapy.

DNA-PKcs kinase activity stabilizes the transcription factor Egr1 in activated immune cells.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is known primarily for its function in DNA double-stranded break repair and nonhomologous end joining (NHEJ). However, DNA-PKcs also has a critical yet undefined role in immunity impacting both myeloid and lymphoid cell lineages spurring interest in targeting DNA-PKcs for therapeutic strategies in immune-related diseases. To gain insight into the function of DNA-PKcs within immune cells, we performed a quantitative phosphoproteomic screen in T cells to identify phosphorylation targets of DNA-PKcs. Our results indicate that DNA-PKcs phosphorylates the transcription factor Egr1 (early growth response protein 1) at serine 301. Expression of Egr1 is induced early upon T cell activation and dictates T cell response by modulating expression of cytokines and key costimulatory molecules such as IL (interleukin) 2, IL6, IFNγ, and NFκB. Inhibition of DNA-PKcs by treatment with a DNA-PKcs specific inhibitor NU7441 or shRNA knockdown increased proteasomal degradation of Egr1. Mutation of serine 301 to alanine via CRISPR-Cas9 reduced EGR1 protein expression and decreased Egr1-dependent transcription of IL2 in activated T cells. Our findings identify DNA-PKcs as a critical intermediary link between T cell activation and T cell fate and a novel phosphosite involved in regulating Egr1 activity.

PTMViz: a tool for analyzing and visualizing histone post translational modification data.

BACKGROUND: Histone post-translational modifications (PTMs) play an important role in our system by regulating the structure of chromatin and therefore contribute to the regulation of gene and protein expression. Irregularities in histone PTMs can lead to a variety of different diseases including various forms of cancer. Histone modifications are analyzed using high resolution mass spectrometry, which generate large amounts of data that requires sophisticated bioinformatics tools for analysis and visualization. PTMViz is designed for downstream differential abundance analysis and visualization of both protein and/or histone modifications. RESULTS: PTMViz provides users with data tables and visualization plots of significantly differentiated proteins and histone PTMs between two sample groups. All the data is packaged into interactive data tables and graphs using the Shiny platform to help the user explore the results in a fast and efficient manner to assess if changes in the system are due to protein abundance changes or epigenetic changes. In the example data provided, we identified several proteins differentially regulated in the dopaminergic pathway between mice treated with methamphetamine compared to a saline control. We also identified histone post-translational modifications including histone H3K9me, H3K27me3, H4K16ac, and that were regulated due to drug exposure. CONCLUSIONS: Histone modifications play an integral role in the regulation of gene expression. PTMViz provides an interactive platform for analyzing proteins and histone post-translational modifications from mass spectrometry data in order to quickly identify differentially expressed proteins and PTMs.

PHF19 promotes multiple myeloma tumorigenicity through PRC2 activation and broad H3K27me3 domain formation.

Dysregulation of polycomb repressive complex 2 (PRC2) promotes oncogenesis partly through its enzymatic function for inducing trimethylation of histone H3 lysine 27 (H3K27me3). However, it remains to be determined how PRC2 activity is regulated in normal and diseased settings. We here report a PRC2-associated cofactor, PHD finger protein 19 (PHF19; also known as polycomb-like 3), as a crucial mediator of tumorigenicity in multiple myeloma (MM). Overexpression and/or genomic amplification of PHF19 is found associated with malignant progression of MM and plasma cell leukemia, correlating to worse treatment outcomes. Using various MM models, we demonstrated a critical requirement of PHF19 for tumor growth in vitro and in vivo. Mechanistically, PHF19-mediated oncogenic effect relies on its PRC2-interacting and chromatin-binding functions. Chromatin immunoprecipitation followed by sequencing profiling showed a critical role for PHF19 in maintaining the H3K27me3 landscape. PHF19 depletion led to loss of broad H3K27me3 domains, possibly due to impaired H3K27me3 spreading from cytosine guanine dinucleotide islands, which is reminiscent to the reported effect of an "onco"-histone mutation, H3K27 to methionine (H3K27M). RNA-sequencing-based transcriptome profiling in MM lines also demonstrated a requirement of PHF19 for optimal silencing of PRC2 targets, which include cell cycle inhibitors and interferon-JAK-STAT signaling genes critically involved in tumor suppression. Correlation studies using patient sample data sets further support a clinical relevance of the PHF19-regulated pathways. Lastly, we show that MM cells are generally sensitive to PRC2 inhibitors. Collectively, this study demonstrates that PHF19 promotes MM tumorigenesis through enhancing H3K27me3 deposition and PRC2's gene-regulatory functions, lending support for PRC2 blockade as a means for MM therapeutics.

Accurate and Sensitive Quantitation of the Dynamic Heat Shock Proteome Using Tandem Mass Tags.

Cells respond to environmental perturbations and insults through modulating protein abundance and function. However, the majority of studies have focused on changes in RNA abundance because quantitative transcriptomics has historically been more facile than quantitative proteomics. Modern Orbitrap mass spectrometers now provide sensitive and deep proteome coverage, allowing direct, global quantification of not only protein abundance but also post-translational modifications (PTMs) that regulate protein activity. We implemented and validated using the well-characterized heat shock response of budding yeast, a tandem mass tagging (TMT), triple-stage mass spectrometry (MS3) strategy to measure global changes in the proteome during the yeast heat shock response over nine time points. We report that basic-pH, ultra-high performance liquid chromatography (UPLC) fractionation of tryptic peptides yields superfractions of minimal redundancy, a crucial requirement for deep coverage and quantification by subsequent LC-MS3. We quantified 2275 proteins across three biological replicates and found that differential expression peaked near 90 min following heat shock (with 868 differentially expressed proteins at 5% false discovery rate). The sensitivity of the approach also allowed us to detect changes in the relative abundance of ubiquitination and phosphorylation PTMs over time. Remarkably, relative quantification of post-translationally modified peptides revealed striking evidence of regulation of the heat shock response by protein PTMs. These data demonstrate that the high precision of TMT-MS3 enables peptide-level quantification of samples, which can reveal important regulation of protein abundance and regulatory PTMs under various experimental conditions.

Serum amyloid P deposition is a sensitive and specific feature of membranous-like glomerulopathy with masked IgG kappa deposits.

Membranous-like glomerulopathy with masked IgG kappa deposits (MGMID) is a recently described pattern of glomerulonephritis with a unique histopathology. The pattern is characterized by subepithelial and/or mesangial immune deposits that are "masked", to immunoglobulin staining by routine immunofluorescence but strongly stain for IgG and kappa light chain after protease digestion. Patients with this pattern of glomerulonephritis are most commonly young females presenting with proteinuria and a vague history of autoimmune disease such as low titer antinuclear antibodies. Here we compared the mass spectrometry profile of laser capture microdissected glomeruli from nine MGMID renal biopsies with eight biopsies showing other patterns of membranous glomerulopathy. The protein most significantly increased in MGMID was serum amyloid P. Immunostaining showed serum amyloid P colocalized with IgG in the glomeruli of MGMID but not with PLA2R-associated membranous glomerulopathy. Serum amyloid P was positive in the glomeruli of all 32 MGMID biopsies but negative in biopsies of other types of membranous glomerulopathies such as those associated with PLA2R and THSD7A. There were four biopsies with glomerular serum amyloid P staining among the 173 biopsies that did not fulfill criteria for MGMID or amyloidosis. All four of these biopsies with positive serum amyloid P staining had a membranous pattern of glomerulopathy with IgG kappa deposits that only differed from MGMID by the lack of "masking". Thus, positive staining within glomerular deposits for serum amyloid P identifies a unique form of glomerulonephritis likely sharing a common pathophysiologic mechanism of disease.

Label-Free Proteomic Approach to Characterize Protease-Dependent and -Independent Effects of sarA Inactivation on the Staphylococcus aureus Exoproteome.

The staphylococcal accessory regulator A ( sarA) impacts the extracellular accumulation of Staphylococcus aureus virulence factors at the level of intracellular production and extracellular protease-mediated degradation. We previously used a proteomics approach that measures protein abundance of all proteoforms to demonstrate that mutation of sarA results in increased levels of extracellular proteases and assesses the impact of this on the accumulation of S. aureus exoproteins. Our previous approach was limited as it did not take into account that large, stable proteolytic products from a given protein could result in false negatives when quantified by total proteoforms. Here, our goal was to use an expanded proteomics approach utilizing a dual quantitative method for measuring abundance at both the total proteoform and full-length exoprotein levels to alleviate these false negatives and thereby provide for characterization of protease-dependent and -independent effects of sarA mutation on the S. aureus exoproteome. Proteins present in conditioned medium from overnight, stationary phase cultures of the USA300 strain LAC, an isogenic sarA mutant, and a sarA mutant unable to produce any of the known extracellular proteases ( sarA/protease) were resolved using one-dimensional gel electrophoresis. Quantitative proteomic comparisons of sarA versus sarA/protease mutants identified proteins that were cleaved in a protease-dependent manner owing to mutation of sarA, and comparisons of sarA/protease mutant versus the LAC parent strain identified proteins in which abundance was altered in a sarA mutant in a protease-independent manner. Furthermore, the proteins uniquely identified by the full-length data analysis approach eliminated false negatives observed in the total proteoform analysis. This expanded approach provided for a more comprehensive analysis of the impact of mutating sarA on the S. aureus exoproteome.

In Vivo Metabolic Tracing Demonstrates the Site-Specific Contribution of Hepatic Ethanol Metabolism to Histone Acetylation.

BACKGROUND: Epigenetic dysregulation through ethanol (EtOH)-induced changes in DNA methylation and histone modifications has been implicated in several alcohol-related disorders such as alcoholic liver disease. EtOH metabolism in the liver results in the formation of acetate, a metabolite that can be converted to acetyl-CoA, which can then be used by histone acetyltransferases to acetylate lysine residues. EtOH metabolism in the liver can also indirectly influence lysine acetylation through NAD+ -dependent sirtuin activity that is altered due to increases in NADH. As a proof-of-concept study to determine the direct influence of hepatic EtOH metabolism on histone acetylation changes, we used heavy-labeled EtOH (13 C2 ) and mass spectrometry (MS) to site specifically characterize lysine acetylation on histone proteins. METHODS: Eight-week-old male C57BL/6J mice were gavaged using a bolus dose of either 13 C2 -labeled EtOH (5 g/kg) or maltose dextrin. Blood and livers were collected at 0, 4, and 24 hours followed by histone protein enrichment and derivatization using acid extraction and propionylation, respectively. Metabolic tracing and relative quantitation of acetylated histone proteins were performed using a hybrid quadrupole-orbitrap mass spectrometer. Data were analyzed using MaxQuant, Xcalibur Qual Browser, and the Bioconductor package "mzR." The contribution of EtOH to histone acetylation was quantified using the change in relative abundance of stable isotope incorporation in acetylated peptides detected by MS. RESULTS: Data show significant incorporation of the EtOH-derived 13 C2 -label into N-terminal lysine acetylation sites on histones H3 and H4 after 4 hours, with rapid turnover of labeled histone acetylation sites and return to endogenous levels at 24 hours postgavage. Moreover, site-specific selectivity was observed in regard to label incorporation into certain lysine acetylation sites as determined by tandem mass spectrometry and comparison to isotope simulations. CONCLUSIONS: These data provide the first quantitative evidence of how hepatic EtOH metabolism directly influences histone lysine acetylation in a site-specific manner and may influence EtOH-induced gene expression through these transcriptionally activating chromatin marks.

Nonsense codon suppression in fission yeast due to mutations of tRNA(Ser.11) and translation release factor Sup35 (eRF3).

In the fission yeast Schizosaccharomyces pombe, sup9 mutations can suppress the termination of translation at nonsense (stop) codons. We localized sup9 physically to the spctrnaser.11 locus and confirmed that one allele (sup9-UGA) alters the anticodon of a serine tRNA. We also found that another purported allele is not allelic. Instead, strains with that suppressor (renamed sup35-F592S) have a single base pair substitution (T1775C) that introduces an amino acid substitution in the Sup35 protein (Sup35-F592S). Reduced functionality of Sup35 (eRF3), the ubiquitous guanine nucleotide-responsive translation release factor of eukaryotes, increases read-through of stop codons. Tetrad dissection revealed that suppression is tightly linked to (inseparable from) the sup35-F592S mutation and that there are no additional extragenic modifiers. The Mendelian inheritance indicates that the Sup35-F592S protein does not adopt an infectious amyloid state ([PSI (+)] prion) to affect suppression, consistent with recent evidence that fission yeast Sup35 does not form prions. We also report that sup9-UGA and sup35-F592S exhibit different strengths of suppression for opal stop codons of ade6-M26 and ade6-M375. We discuss possible mechanisms for the variation in suppressibility exhibited by the two alleles.

Rapid, efficient and precise allele replacement in the fission yeast Schizosaccharomyces pombe.

Gene targeting provides a powerful tool to modify endogenous loci to contain specific mutations, insertions and deletions. Precise allele replacement, with no other chromosomal changes (e.g., insertion of selectable markers or heterologous promoters), maintains physiologically relevant context. Established methods for precise allele replacement in fission yeast employ two successive rounds of transformation and homologous recombination and require genotyping at each step. The relative efficiency of homologous recombination is low and a high rate of false positives during the second round of gene targeting further complicates matters. We report that pop-in, pop-out allele replacement circumvents these problems. We present data for 39 different allele replacements, involving simple and complex modifications at seven different target loci, that illustrate the power and utility of the approach. We also developed and validated a rapid, efficient process for precise allele replacement that requires only one round each of transformation and genotyping. We show that this process can be applied in population scale to an individual target locus, without genotyping, to identify clones with an altered phenotype (targeted forward genetics). It is therefore suitable for saturating, in situ, locus-specific mutation screens (e.g., of essential or non-essential genes and regulatory DNA elements) within normal chromosomal context.

Characterization of methionine dependence in melanoma cells.

Dietary methionine restriction is associated with a reduction in tumor growth in preclinical studies and an increase in lifespan in animal models. The mechanism by which methionine restriction inhibits tumor growth while sparing normal cells is incompletely understood. We do know that normal cells can utilize methionine or homocysteine interchangeably (methionine independence) while most cancer cells are strictly dependent on methionine availability. Here, we compared a typical methionine dependent and a rare methionine independent melanoma cell line. We show that replacing methionine, a methyl donor, with its precursor homocysteine generally induced hypomethylation in gene promoters. This decrease was similar in methionine dependent and methionine independent cells. There was only a low level of pathway enrichment, suggesting that the hypomethylation is generalized rather than gene specific. Whole proteome and transcriptome were also analyzed. This analysis revealed that contrarily to the effect on methylation, the replacement of methionine with homocysteine had a much greater effect on the transcriptome and proteome of methionine dependent cells than methionine independent cells. Interestingly, methionine adenosyltransferase 2A (MAT2A), responsible for the synthesis of S-adenosylmethionine from methionine, was equally strongly upregulated in both cell lines. This suggests that the absence of methionine is equally detected but triggers different outcomes in methionine dependent versus independent cells. Our analysis reveals the importance of cell cycle control, DNA damage repair, translation, nutrient sensing, oxidative stress and immune functions in the cellular response to methionine stress in melanoma.

Bifunctional Inhibitor Reveals NEK2 as a Therapeutic Target and Regulator of Oncogenic Pathways in Lymphoma.

Expression of the serine/threonine kinase never in mitosis gene A (NIMA)-related kinase 2 (NEK2) is essential for entry into mitosis via its role in facilitating centrosome separation. Its overactivity can lead to tumorigenesis and drug resistance through the activation of several oncogenic pathways, including AKT. Although the cancer-enabling activities of NEK2 are documented in many malignancies, including correlations with poor survival in myeloma, breast, and non-small cell lung cancer, little is known about the role of NEK2 in lymphoma. Here, in tumors from patients with diffuse large B-cell lymphoma (DLBCL), the most common, aggressive non-Hodgkin lymphoma, we found a high abundance of NEK2 mRNA and protein associated with an inferior overall survival. Using our recently developed NEK2 inhibitor, NBI-961, we discovered that DLBCL cell lines and patient-derived cells exhibit a dependency on NEK2 for their viability. This compromised cell fitness was directly attributable to efficient NEK2 inhibition and proteasomal degradation by NBI-961. In a subset of particularly sensitive DLBCL cells, NBI-961 induced G2/mitosis arrest and apoptosis. In contrast, an existing indirect NEK2 inhibitor, INH154, did not prevent NEK2 autophosphorylation, induce NEK2 proteasomal degradation, or affect cell viability. Global proteomics and phospho-proteomics revealed that NEK2 orchestrates cell-cycle and apoptotic pathways through regulation of both known and new signaling molecules. We show the loss of NEK2-sensitized DLBCL to the chemotherapy agents, doxorubicin and vincristine, and effectively suppressed tumor growth in mice. These studies establish the oncogenic activity of NEK2 in DLBCL and set the foundation for development of anti-NEK2 therapeutic strategies in this frequently refractory and relapse-prone cancer.

Characterization of methionine dependence in melanoma cells.

Dietary methionine restriction is associated with a reduction in tumor growth in preclinical studies and an increase in lifespan in animal models. The mechanism by which methionine restriction inhibits tumor growth while sparing normal cells is incompletely understood. We do know that normal cells can utilize methionine or homocysteine interchangeably (methionine independence) while most cancer cells are strictly dependent on methionine availability. Here, we compared a typical methionine dependent and a rare methionine independent melanoma cell line. We show that replacing methionine, a methyl donor, with its precursor homocysteine generally induced hypomethylation in gene promoters. This decrease was similar in methionine dependent and methionine independent cells. There was only a low level of pathway enrichment, suggesting that the hypomethylation is generalized rather than gene specific. Whole proteome and transcriptome were also analyzed. This analysis revealed that contrarily to the effect on methylation, the replacement of methionine with homocysteine had a much greater effect on the transcriptome and proteome of methionine dependent cells than methionine independent cells. Interestingly, methionine adenosyltransferase 2A (MAT2A), responsible for the synthesis of s-adenosylmethionine from methionine, was equally strongly upregulated in both cell lines. This suggests that the absence of methionine is equally detected but triggers different outcomes in methionine dependent versus independent cells. Our analysis reveals the importance of cell cycle control, DNA damage repair, translation, nutrient sensing, oxidative stress and immune functions in the cellular response to methionine stress in melanoma.

The Alternative Pathway Is Necessary and Sufficient for Complement Activation by Anti-THSD7A Autoantibodies, Which Are Predominantly IgG4 in Membranous Nephropathy.

Membranous nephropathy (MN) is an immune kidney disease characterized by glomerular subepithelial immune complexes (ICs) containing antigen, IgG, and products of complement activation. Whereas proteinuria is caused by complement-mediated podocyte injury, the pathways of complement activation remain controversial due to the predominance of IgG4 in ICs, an IgG subclass considered unable to activate complement. THSD7A, a transmembrane protein expressed on podocytes, is the target autoantigen in ~3% of cases of primary MN. In this study, we analyzed sera from 16 patients with THSD7A-associated MN with regard to the anti-THSD7A IgG subclasses and their ability to fix complement in vitro. The serum concentration of anti-THSD7A IgG varied over two orders of magnitude (1.3-243 µg/mL). As a relative proportion of all IgG anti-THSD7A, IgG4 was by far the most abundant subclass (median 79%), followed by IgG1 (median 11%). IgG4 was the dominant subclass of anti-THSD7A antibodies in 14 sera, while IgG1 was dominant in one and co-dominant in another. One quarter of MN sera additionally contained low levels of anti-THSD7A IgA1. ICs formed by predominantly IgG4 anti-THSD7A autoantibodies with immobilized THSD7A were relatively weak activators of complement in vitro, compared to human IgG1 and IgG3 mAbs used as positive control. Complement deposition on THSD7A ICs was dose-dependent and occurred to a significant extent only at relatively high concentration of anti-THSD7A IgG. C3b fixation by THSD7A ICs was completely abolished in factor B-depleted sera, partially inhibited in C4-depleted sera, unchanged in C1q-depleted sera, and also occurred in Mg-EGTA buffer. These results imply that THSD7A ICs predominantly containing IgG4 activate complement at high IgG4 density, which strictly requires a functional alternative pathway, whereas the classical and lectin pathways are dispensable. These findings advance our understanding of how IgG4 antibodies activate complement.

Discovery of a dual WDR5 and Ikaros PROTAC degrader as an anti-cancer therapeutic.

WD repeat domain 5 (WDR5), an integral component of the MLL/KMT2A lysine methyltransferase complex, is critically involved in oncogenesis and represents an attractive onco-target. Inhibitors targeting protein-protein interactions (PPIs) between WDR5 and its binding partners, however, do not inhibit all of WDR5-mediated oncogenic functions and exert rather limited antitumor effects. Here, we report a cereblon (CRBN)-recruiting proteolysis targeting chimera (PROTAC) of WDR5, MS40, which selectively degrades WDR5 and the well-established neo-substrates of immunomodulatory drugs (IMiDs):CRBN, the Ikaros zinc finger (IKZF) transcription factors IKZF1 and IKZF3. MS40-induced WDR5 degradation caused disassociation of the MLL/KMT2A complex off chromatin, resulting in decreased H3K4me2. Transcriptomic profiling revealed that targets of both WDR5 and IMiDs:CRBN were significantly repressed by treatment of MS40. In MLL-rearranged leukemias, which exhibit IKZF1 high expression and dependency, co-suppression of WDR5 and Ikaros by MS40 is superior in suppressing oncogenesis to the WDR5 PPI inhibitor, to MS40's non-PROTAC analog controls (MS40N1 and MS40N2, which do not bind CRBN and WDR5, respectively), and to a matched VHL-based WDR5 PROTAC (MS169, which degrades WDR5 but not Ikaros). MS40 suppressed the growth of primary leukemia patient cells in vitro and patient-derived xenografts in vivo. Thus, dual degradation of WDR5 and Ikaros is a promising anti-cancer strategy.