An inhibitor of KDM5 demethylases reduces survival of drug-tolerant cancer cells.

The KDM5 family of histone demethylases catalyzes the demethylation of histone H3 on lysine 4 (H3K4) and is required for the survival of drug-tolerant persister cancer cells (DTPs). Here we report the discovery and characterization of the specific KDM5 inhibitor CPI-455. The crystal structure of KDM5A revealed the mechanism of inhibition of CPI-455 as well as the topological arrangements of protein domains that influence substrate binding. CPI-455 mediated KDM5 inhibition, elevated global levels of H3K4 trimethylation (H3K4me3) and decreased the number of DTPs in multiple cancer cell line models treated with standard chemotherapy or targeted agents. These findings show that pretreatment of cancer cells with a KDM5-specific inhibitor results in the ablation of a subpopulation of cancer cells that can serve as the founders for therapeutic relapse.

Cell death induced by the Jak2 inhibitor, G6, correlates with cleavage of vimentin filaments.

Hyperkinetic Jak2 tyrosine kinase signaling has been implicated in several human diseases including leukemia, lymphoma, myeloma, and the myeloproliferative neoplasms. Using structure-based virtual screening, we previously identified a novel Jak2 inhibitor named G6. We showed that G6 specifically inhibits Jak2 kinase activity and suppresses Jak2-mediated cellular proliferation. To elucidate the molecular and biochemical mechanisms by which G6 inhibits Jak2-mediated cellular proliferation, we treated Jak2-V617F expressing human erythroleukemia (HEL) cells for 12 h with either vehicle control or 25 µM of the drug and compared protein expression profiles using two-dimensional gel electrophoresis. One differentially expressed protein identified by electrospray mass spectroscopy was the intermediate filament protein, vimentin. It was present in DMSO treated cells but absent in G6 treated cells. HEL cells treated with G6 showed both time- and dose-dependent cleavage of vimentin as well as a marked reorganization of vimentin intermediate filaments within intact cells. In a mouse model of Jak2-V617F mediated human erythroleukemia, G6 also decreased the levels of vimentin protein, in vivo. The G6-induced cleavage of vimentin was found to be Jak2-dependent and calpain-mediated. Furthermore, we found that intracellular calcium mobilization is essential and sufficient for the cleavage of vimentin. Finally, we show that the cleavage of vimentin intermediate filaments, per se, is sufficient to reduce HEL cell viability. Collectively, these results suggest that G6-induced inhibition of Jak2-mediated pathogenic cell growth is concomitant with the disruption of intracellular vimentin filaments. As such, this work describes a novel pathway for the targeting of Jak2-mediated pathological cell growth.

Activation domain-dependent degradation of somatic Wee1 kinase.

Cell cycle progression is dependent upon coordinate regulation of kinase and proteolytic pathways. Inhibitors of cell cycle transitions are degraded to allow progression into the subsequent cell cycle phase. For example, the tyrosine kinase and Cdk1 inhibitor Wee1 is degraded during G(2) and mitosis to allow mitotic progression. Previous studies suggested that the N terminus of Wee1 directs Wee1 destruction. Using a chemical mutagenesis strategy, we report that multiple regions of Wee1 control its destruction. Most notably, we find that the activation domain of the Wee1 kinase is also required for its degradation. Mutations in this domain inhibit Wee1 degradation in somatic cell extracts and in cells without affecting the overall Wee1 structure or kinase activity. More broadly, these findings suggest that kinase activation domains may be previously unappreciated sites of recognition by the ubiquitin proteasome pathway.

Sperm proteomics reveals intensified selection on mouse sperm membrane and acrosome genes.

Spermatozoa are a focal point for the impact of sexual selection due to sperm competition and sperm-female interactions in a wide range of sexually reproducing organisms. In-depth molecular investigation of the ramifications of these selective regimes has been limited due to a lack of information concerning the molecular composition of sperm. In this study, we utilize three previously published proteomic data sets in conjunction with our whole mouse sperm proteomic analysis to delineate cellular regions of sperm most impacted by positive selection. Interspecific analysis reveals robust evolutionary acceleration of sperm cell membrane genes (which include genes encoding acrosomal and sperm cell surface proteins) relative to other sperm genes, and evidence for positive selection in approximately 22% of sperm cell membrane components was obtained using maximum likelihood models. The selective forces driving the accelerated evolution of these membrane proteins may occur at a number of locations during sperm development, maturation, and transit through the female reproductive tract where the sperm cell membrane and eventually the acrosome are exposed to the extracellular milieu and available for direct cell-cell interactions.

A coactivator trap identifies NONO (p54nrb) as a component of the cAMP-signaling pathway.

Signal transduction pathways often use a transcriptional component to mediate adaptive cellular responses. Coactivator proteins function prominently in these pathways as the conduit to the basic transcriptional machinery. Here we present a high-throughput cell-based screening strategy, termed the "coactivator trap," to study the functional interactions of coactivators with transcription factors. We applied this strategy to the cAMP signaling pathway, which utilizes two families of coactivators, the cAMP response element binding protein (CREB) binding protein (CBP)/p300 family and the recently identified transducers of regulated CREB activity family (TORCs1-3). In addition to identifying numerous known interactions of these coactivators, this analysis identified NONO (p54(nrb)) as a TORC-interacting protein. RNA interference experiments demonstrate that NONO is necessary for cAMP-dependent activation of CREB target genes in vivo. Furthermore, TORC2 and NONO complex on cAMP-responsive promoters, and NONO acts as a bridge between the CREB/TORC complex and RNA polymerase II. These data demonstrate the utility of the coactivator trap by identification of a component of cAMP-mediated transcription.

Mapping the phosphorylation sites of Ulk1.

Ulk1 is a serine/threonine kinase that controls macroautophagy, an essential homeostatic recycling pathway that degrades bulk cytoplasmic material and directs the turnover of organelles such as peroxisomes and mitochondria. Further, macroautophagy is potently induced by signals that trigger metabolic stress, such as hypoxia and amino acid starvation, where its recycling functions provide macromolecules necessary to maintain catabolic metabolism and cell survival. Substrates for Ulk1 have not been identified, and little is known regarding post-translational control of Ulk1 kinase activity and function. To gain insights into the regulatory mechanisms of Ulk1, we developed a robust purification protocol for Ulk1 and demonstrated that Ulk1 is highly phosphorylated and requires autophosphorylation for stability. Importantly, high-resolution, tandem mass spectrometry identified multiple sites of phosphorylation on Ulk1, including several within domains known to regulate macroautophagy. Differential phosphorylation analyses also identified sites of phosphorylation in the C-terminal domain that depend upon or require Ulk1 autophosphorylation.

Strategies to recover proteins from ocular tissues for proteomics.

We present here the results of protein extraction from different ocular regions using different detergents. Extraction strategies used to determine optimal protein extraction included: pressure cycling and aqueous-organic phase extraction in combination with electrophoretic fractionation for anterior, posterior, and peripapillary sclera. Detergent extraction of proteins from freshly enucleated porcine eyes (n = 8) showed significant differences for different eye regions. Protein yield ranged from 2.3 to 50.7 mug protein/mg for different ocular tissues, with the lens yielding the most protein. ASB-14 and Triton X-100 provided the best protein yields (n = 10) for anterior and posterior sclera. The spectrophotometric measurements for ASB-14 were not consistent with SDS-PAGE densitometry. A combination of 0.5% Triton X-100, 0.5% Tween-20, and 0.1% Genapol C-100 was found optimal for extraction from sclera. Proteins from different regions of the eye are best extracted with different detergents. The pressure cycling technology provided superior extraction compared to the other methods. Additional aqueous-organic phase partitioning enables superior fractionation when compared to SDS-PAGE alone. Organic phase fractionation is compatible with MS and allowed identification of 34, 71, and 77 proteins respectively from anterior, posterior, and peripapillary sclera. The extraction strategy may affect the final outcome in protein profiling by MS or by other methods.

The N-terminal SH2 domain of the tyrosine phosphatase, SHP-2, is essential for Jak2-dependent signaling via the angiotensin II type AT1 receptor.

Previous work has suggested that the protein tyrosine phosphatase, SHP-2, may act to facilitate angiotensin II (Ang II)-mediated, Jak2-dependent signaling. However, the mechanisms by which this occurs are not known. Here, Ang II-mediated, Jak2-dependent signaling was analyzed in a fibroblast cell line lacking the N-terminal, SH2 domain of SHP-2 (SHP-2(Delta46-110)). While the SHP-2(Delta46-110) cells were capable of activating Jak2 tyrosine kinase, they were unable to facilitate AT1 receptor/Jak2 co-association, STAT activation and subsequent Ang II-mediated gene transcription when compared to wild type control cells. These data therefore suggested that the N-terminal SH2 domain of SHP-2 was acting to recruit Jak2 to the AT1 receptor signaling complex. We found that the N-terminal SH2 domain of SHP-2 binds Jak2 predominantly, but not exclusively at tyrosine 201. Mass spectrometry analysis confirmed that this tyrosine residue is in fact phosphorylated. When this tyrosine was converted to phenylalanine, the ability of Jak2 to activate subsequent downstream signaling events was reduced. In summary, we have identified a novel site of Jak2 tyrosine autophosphorylation; namely, tyrosine 201. Our data suggest that the N-terminal SH2 domain of SHP-2 binds this amino acid residue. The functional consequence of this interaction is to recruit Jak2 to the AT1 receptor signaling complex and in turn promote downstream Jak2-dependent signaling.

Deep learning using tumor HLA peptide mass spectrometry datasets improves neoantigen identification.

Neoantigens, which are expressed on tumor cells, are one of the main targets of an effective antitumor T-cell response. Cancer immunotherapies to target neoantigens are of growing interest and are in early human trials, but methods to identify neoantigens either require invasive or difficult-to-obtain clinical specimens, require the screening of hundreds to thousands of synthetic peptides or tandem minigenes, or are only relevant to specific human leukocyte antigen (HLA) alleles. We apply deep learning to a large (N = 74 patients) HLA peptide and genomic dataset from various human tumors to create a computational model of antigen presentation for neoantigen prediction. We show that our model, named EDGE, increases the positive predictive value of HLA antigen prediction by up to ninefold. We apply EDGE to enable identification of neoantigens and neoantigen-reactive T cells using routine clinical specimens and small numbers of synthetic peptides for most common HLA alleles. EDGE could enable an improved ability to develop neoantigen-targeted immunotherapies for cancer patients.

The response of bovine spermatozoa to bicarbonate and its use to assess the influence of added oviductal epithelial proteins on cryopreservation.

The oviduct is a crucial organ for fertilization and has been demonstrated to perform a variety of interactions with spermatozoa ranging from sperm storage, to stabilizing sperm membranes and reducing free radicals. The oviduct is separated into 2 anatomically and physiologically distinct regions: the isthmus, in which sperm are stored, and the ampulla where fertilization occurs. We aimed to investigate whether proteins derived from different regions of the bovine oviduct had beneficial effects on bovine sperm membrane integrity, osmotic resistance, and motility following cryopreservation. The extent to which sperm motility could be activated by bicarbonate was demonstrated and used as a novel approach to postthaw sperm assessment. While oviductal proteins did not increase the degree of postthaw sperm viability, spermatozoa exposed to the isthmic proteins before freezing showed higher osmotic resistance after thawing. The presence of bicarbonate increased the proportion of spermatozoa with high curvilinear (VCL) and straight line velocity (VSL) in all treatment groups. After thawing, spermatozoa exposed to isthmic proteins had higher VCL and VSL than spermatozoa exposed to the ampullar proteins. We conclude that proteins derived from the isthmus can stabilize and protect spermatozoa during cryopreservation.

A cell based screening approach for identifying protein degradation regulators.

Cellular transitions are achieved by the concerted actions of regulated degradation pathways. In the case of the cell cycle, ubiquitin mediated degradation ensures unidirectional transition from one phase to another. For instance, turnover of the cell cycle regulator cyclin B1 occurs after metaphase to induce mitotic exit. To better understand pathways controlling cyclin B1 turnover, the N-terminal domain of cyclin B1 was fused to luciferase to generate an N-cyclin B1-luciferase protein that can be used as a reporter for protein turnover. Prior studies demonstrated that cell-based screens using this reporter identified small molecules inhibiting the ubiquitin ligase controlling cyclin B1-turnover. Our group adapted this approach for the G2-M regulator Wee1 where a Wee1-luciferase construct was used to identify selective small molecules inhibiting an upstream kinase that controls Wee1 turnover. In the present study we present a screening approach where cell cycle regulators are fused to luciferase and overexpressed with cDNAs to identify specific regulators of protein turnover. We overexpressed approximately 14,000 cDNAs with the N-cyclin B1-luciferase fusion protein and determined its steady-state level relative to other luciferase fusion proteins. We identified the known APC/C regulator Cdh1 and the F-box protein Fbxl15 as specific modulators of N-cyclin B1-luciferase steady-state levels and turnover. Collectively, our studies suggest that analyzing the steady-state levels of luciferase fusion proteins in parallel facilitates identification of specific regulators of protein turnover.

An Alternative Approach to ChIP-Seq Normalization Enables Detection of Genome-Wide Changes in Histone H3 Lysine 27 Trimethylation upon EZH2 Inhibition.

Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) has been instrumental in inferring the roles of histone post-translational modifications in the regulation of transcription, chromatin compaction and other cellular processes that require modulation of chromatin structure. However, analysis of ChIP-seq data is challenging when the manipulation of a chromatin-modifying enzyme significantly affects global levels of histone post-translational modifications. For example, small molecule inhibition of the methyltransferase EZH2 reduces global levels of histone H3 lysine 27 trimethylation (H3K27me3). However, standard ChIP-seq normalization and analysis methods fail to detect a decrease upon EZH2 inhibitor treatment. We overcome this challenge by employing an alternative normalization approach that is based on the addition of Drosophila melanogaster chromatin and a D. melanogaster-specific antibody into standard ChIP reactions. Specifically, the use of an antibody that exclusively recognizes the D. melanogaster histone variant H2Av enables precipitation of D. melanogaster chromatin as a minor fraction of the total ChIP DNA. The D. melanogaster ChIP-seq tags are used to normalize the human ChIP-seq data from DMSO and EZH2 inhibitor-treated samples. Employing this strategy, a substantial reduction in H3K27me3 signal is now observed in ChIP-seq data from EZH2 inhibitor treated samples.

EZH2 inhibitor efficacy in non-Hodgkin's lymphoma does not require suppression of H3K27 monomethylation.

The histone lysine methyltransferase (MT) Enhancer of Zeste Homolog 2 (EZH2) is considered an oncogenic driver in a subset of germinal center B-cell-like diffuse large B cell lymphoma (GCB-DLBCL) and follicular lymphoma due to the presence of recurrent, monoallelic mutations in the EZH2 catalytic domain. These genomic data suggest that targeting the EZH2 MT activity is a valid therapeutic strategy for the treatment of lymphoma patients with EZH2 mutations. Here we report the identification of highly potent and selective EZH2 small molecule inhibitors, their validation by a cellular thermal shift assay, application across a large cell panel representing various non-Hodgkin's lymphoma (NHL) subtypes, and their efficacy in EZH2mutant-containing GCB-DLBCL xenograft models. Surprisingly, our EZH2 inhibitors selectively affect the turnover of trimethylated, but not monomethylated histone H3 lysine 27 at pharmacologically relevant doses. Importantly, we find that these inhibitors are broadly efficacious also in NHL models with wild-type EZH2.

Direct interaction between AR and PAK6 in androgen-stimulated PAK6 activation.

A p21-activated kinase 6 (PAK6) was previously identified to be an androgen receptor (AR) interacting protein through a yeast two-hybrid screening. We used hormone responsive prostate cancer LAPC4 and LNCap cell lines as models to study the signaling events associated with androgen stimulation and PAK6. An androgen-stimulated PAK6 kinase activation was observed in LAPC4 cells expressing endogenous PAK6 and in LNCap cells ectopically expressing a wild type PAK6. This activation was likely mediated through a direct interaction between AR and PAK6 since siRNA knock-down of AR in LAPC4 cells downregulated androgen-stimulated PAK6 activation. In addition, LNCap cells expressing a non-AR-interacting PAK6 mutant exhibited dampened androgen-stimulated kinase activation. As a consequence of androgen-stimulated activation, PAK6 was phosphorylated at multiple serine/threonine residues including the AR-interacting domain of PAK6. Furthermore, androgen-stimulation promoted prostate cancer cell motility and invasion were demonstrated in LNCap cells ectopically expressing PAK6-WT. In contrast, LNCap expressing non-AR-interacting mutant PAK6 did not respond to androgen stimulation with increased cell motility and invasion. Our results demonstrate that androgen-stimulated PAK6 activation is mediated through a direct interaction between AR and PAK6 and PAK6 activation promotes prostate cancer cells motility and invasion.

Identification of EZH2 and EZH1 small molecule inhibitors with selective impact on diffuse large B cell lymphoma cell growth.

The histone methyltransferase enhancer of Zeste homolog 2 (EZH2) is a candidate oncogene due to its prevalent overexpression in malignant diseases, including late stage prostate and breast cancers. The dependency of cancer cells on EZH2 activity is also predicated by recurrent missense mutations residing in the catalytic domain of EZH2 that have been identified in subtypes of diffuse large B cell lymphoma, follicular lymphoma and melanoma. Herein, we report the identification of a highly selective small molecule inhibitor series of EZH2 and EZH1. These compounds inhibit wild-type and mutant versions of EZH2 with nanomolar potency, suppress global histone H3-lysine 27 methylation, affect gene expression, and cause selective proliferation defects. These compounds represent a structurally distinct EZH2 inhibitor chemotype for the exploration of the role of Polycomb Repressive Complex 2-mediated H3K27 methylation in various biological contexts.

Phosphorylation of Y372 is critical for Jak2 tyrosine kinase activation.

Jak2 tyrosine kinase plays an important role in cytokine mediated signal transduction. There are 49 tyrosine residues in Jak2 and phosphorylation of some of these are known to play important roles in the regulation of Jak2 kinase activity. Here, using mass spectrometry, we identified tyrosine residues Y372 and Y373 as novel sites of Jak2 phosphorylation. Mutation of Y372 to F (Y372F) significantly inhibited Jak2 phosphorylation, including that of Y1007, whereas the Jak2-Y373F mutant displayed only modest reduction in phosphorylation. Relative to Jak2-WT, the ability of Jak2-Y372F to bind to and phosphorylate STAT1 was decreased, resulting in reduced Jak2-mediated downstream gene transcription. While the Y372F mutation had no effect on receptor-independent, hydrogen peroxide-mediated Jak2 activation, it impaired interferon-gamma (IFNγ) and epidermal growth factor (EGF)-dependent Jak2 activation. Interestingly however, the Y372F mutant exhibited normal receptor binding properties. Finally, co-expression of SH2-Bß only partially restored the activation of the Jak2-Y372F mutant suggesting that the mechanism whereby phosphorylation of Y372 is important for Jak2 activation is via dimerization. As such, our results indicate that Y372 plays a critical yet differential role in Jak2 activation and function via a mechanism involving Jak2 dimerization and stabilization of the active conformation.

Interaction between TAK1-TAB1-TAB2 and RCAN1-calcineurin defines a signalling nodal control point.

The calcium-activated protein phosphatase calcineurin is controlled by regulator of calcineurin (RCAN) in organisms ranging from yeast to mammals. Here we performed a yeast two-hybrid screen with RCAN1 as bait, identifying TAK1 binding protein 2 (TAB2) as an interacting partner. TAB2 interacted directly with RCAN1 in vitro and in vivo, recruiting TAK1, TAB1 and calcineurin, forming a macromolecular signalling complex. Overexpression of TAK1 and TAB1, or active TAK1(DeltaN), promoted direct phosphorylation of RCAN1 in vitro and in vivo. TAK1 phosphorylated RCAN1 at Ser 94 and Ser 136, converting RCAN1 from an inhibitor to a facilitator of calcineurin-NFAT signalling, and enhancing NFATc1 nuclear translocation, NFAT transcriptional activation and the hypertrophic growth of cultured cardiomyocytes. The TAK1-TAB1-TAB2 and the calcineurin-NFAT signalling modules did not interact in Rcan1/2- or Tab2-deficient mouse embryonic fibroblast (MEF) cultures. Calcineurin activation also dephosphorylated and inhibited TAK1 and TAB1, an effect that was absent in Rcan1/2 deficient MEFs. Functionally, TAK1 was indispensable for the cardiomyocyte growth response induced by pro-hypertrophic stimuli through calcineurin. These results describe a signalling relationship between two central regulatory pathways in which TAK1-TAB1-TAB2 selectively induces calcineurin-NFAT signalling through direct phosphorylation of RCAN1, while calcineurin activation diminishes TAK1 signalling by dephosphorylation of TAK1 and TAB1.

Epidermal growth factor receptor variant III-induced glioma invasion is mediated through myristoylated alanine-rich protein kinase C substrate overexpression.

Glioblastoma multiforme (GBM) is the most common and most malignant adult brain tumor. A characteristic of GBM is their highly invasive nature, making complete surgical resection impossible. The most common gain-of-function alteration in GBM is amplification, overexpression, and mutations of the epidermal growth factor receptor (EGFR). The constitutively activated mutant EGFR variant III (EGFRvIII), found in approximately 20% of GBM, confers proliferative and invasive advantage. The signaling cascades downstream of aberrant EGFR activation contributing to the invasive phenotype are not completely understood. Here, we show myristoylated alanine-rich protein kinase C substrate (MARCKS), previously implicated in cell adhesion and motility, contributes to EGFR-mediated invasion of human GBM cells. EGFRvIII-expressing or EGF-stimulated human GBM cells increased expression, phosphorylation, and cytosolic translocation of MARCKS in a protein kinase C-alpha-dependent manner. Down-regulation of MARCKS expression with small interfering RNA in GBM cells expressing EGFRvIII led to decreased cell adhesion, spreading, and invasion. Elucidation of mechanisms that promote EGFRvIII-mediated tumorigenesis in GBM, such as MARCKS, provides additional understanding and potential biological targets against this currently terminal human cancer.

Proteomics of the nucleus ovoidalis and field L brain regions of zebra finch.

The purpose of present study is to analyze the brain proteome of the nucleus ovoidalis (OV) and Field L regions of the zebra finch (Taeniopygia guttata). The OV and Field L are important brain nuclei in song learning in zebra finches; their analyses identified a total of 79 proteins. The zebra finch brain proteome analyses are poised to provide clues about cell and circuit layout as well as possible circuit function.

Proteomic analyses of songbird (Zebra finch; Taeniopygia guttata) retina.

Proteomic analyses of male songbird (Zebra finch; Taeniopygia guttata; ZF) retina were performed resulting in identification of 129 proteins. Comparison of T. guttata retinal proteome with that of chicken found proteins detected in both retinas. Immunohistochemical analyses of T. guttata retinal sections and Western analyses of total retinal protein extract were performed confirming presence of select bona fide retinal proteins. Results demonstrate the utility of one-dimensional gel fractionation for mass spectrometry and will be useful for future proteomic comparison of songbird retina and brain tissues in different behavioral and pharmacological studies.