Immunoprecipitation Mass Spectrometry Research Articles

Fucosyltransferase 4 Shapes Oncogenic Glycoproteome to Drive Metastasis of Lung Adenocarcinoma

Background: Aberrant fucosylation plays a critical role in lung cancer progression. Nevertheless, the key fucosyltransferase with prognostic significance in lung cancer patients, the enzyme’s intracellular targets and complex molecular mechanisms underlying lung cancer metastasis remain incompletely understood. Methods: We performed a large-scale transcriptome-clinical correlation to identify major fucosyltransferases with significant prognostic values. Invasion, migration, cell adhesion assays were performed using lung cancer cells subject to genetic manipulation of FUT4 levels. Genome-wide RNA-seq and immunoprecipitation-mass spectrometry were used to characterize major cellular processes driven by FUT4, as well as profiling its intracellular protein targets. We also performed lung homing and metastasis assays in mouse xenograft models to determine in vivo phenotypes of high FUT4-expressing cancer cells. Findings: We show that FUT4 is associated with poor overall survival in lung adenocarcinoma patients. High FUT4 expression promotes lung cancer invasion, migration, epithelial-to-mesenchymal transition and cell adhesion. FUT4-mediated aberrant fucosylation markedly activates multiple cellular processes, including membrane trafficking, cell cycle, and major oncogenic signaling pathways. The pathway augmentation effects are independent of receptor tyrosine kinase mutations. Notably, genetic depletion of FUT4 or targeting FUT4-driven pathways diminishes lung colonization and distant metastases of lung cancer cells in mouse xenograft models. Interpretation: We propose that FUT4 can be a prognostic predictor and therapeutic target in lung cancer metastasis. Our data also offer a potential therapeutic strategy using targeted therapy in a subset of patients with high FUT4-expressing tumors with no targetable mutations. Funding Statement: Research supported by grants from Ministry of Science and Technology (MOST 107-2314-B-002 -241, MOST 105-2628-B-002 -040), and National Health Research Institutes (NHRIEX106-10610BC) in Taiwan. Declaration of Interests: The authors have no conflicts of interest to declare. Ethics Approval Statement: The study on primary tumor tissues from lung cancer patients was approved by the Institutional Review Board (IRB) of National Taiwan University Hospital (Approval No.: 201701010RINB). Written informed consent was obtained from all participants prior to inclusion in the study. Every participant was assigned a number and the data were analyzed at the group level for patient de-identification. All mice experiments were performed in compliance with an experimental protocol (20160126) approved by the NTU College of Medicine Institutional Animal Care and Use Committee (IACUC).

FGA isoform as an indicator of targeted therapy for EGFR mutated lung adenocarcinoma.

Epidermal growth factor receptor (EGFR) gene is frequently mutated in non-small cell lung cancer (NSCLC), which can be targeted by EGFR tyrosine kinase inhibitors (TKIs). It is hard, however, to monitor the performance of EGFR-TKI therapy dynamically. Therefore, therapeutic indicators are urgently needed. Novel antibody microarray, containing 41,472 antibodies, was used for comprehensive analyzing of serum samples from 9 normal subjects and 9 EGFR mutated lung adenocarcinoma patients at three EGFR-TKI treatment time points, including before treatment (Baseline), partial response (PR) during treatment, and disease progression (PD) after resistance. Through microarray data analysis, five candidate antibodies were screened out for confirmation in serum samples and the verified one was utilized for candidate protein identification through immunoprecipitation-mass spectrometry strategy. A novel protein, isoform 2 of fibrinogen alpha chain (FGA2), was revealed and verified in the discovery sample set. Its performance as therapy indicator was further evaluated in another pre-validation sample set (n = 60). Our data confirmed that serum FGA2 level was correlated with EGFR-TKI response (p < 0.05). The expression and secretion of FGA2 in hepatocytes were inhibited by EGFR-TKI, partially explaining the downregulation of FGA2 in serum. Our results demonstrate that FGA2 is an indicator of targeted therapy for EGFR mutated lung adenocarcinoma. KEY MESSAGES: Antibody microarray was coupled with mass spectrometry for proteomics research. FGA2 was discovered as an indicator of EGFR-TKI targeted therapy. FGA2's expression/secretion in hepatocytes was dramatically inhibited by EGFR-TKI.

TFIIIC Binding to Alu Elements Controls Gene Expression via Chromatin Looping and Histone Acetylation.

SummaryHow repetitive elements, epigenetic modifications, and architectural proteins interact ensuring proper genome expression remains poorly understood. Here, we report regulatory mechanisms unveiling a central role of Alu elements (AEs) and RNA polymerase III transcription factor C (TFIIIC) in structurally and functionally modulating the genome via chromatin looping and histone acetylation. Upon serum deprivation, a subset of AEs pre-marked by the activity-dependent neuroprotector homeobox Protein (ADNP) and located near cell-cycle genes recruits TFIIIC, which alters their chromatin accessibility by direct acetylation of histone H3 lysine-18 (H3K18). This facilitates the contacts of AEs with distant CTCF sites near promoter of other cell-cycle genes, which also become hyperacetylated at H3K18. These changes ensure basal transcription of cell-cycle genes and are critical for their re-activation upon serum re-exposure. Our study reveals how direct manipulation of the epigenetic state of AEs by a general transcription factor regulates 3D genome folding and expression.

Qip-Mass Spectrometry in High Risk Smoldering Multiple Myeloma Patients Included in the GEM-CESAR Trial: Comparison with Conventional and Minimal Residual Disease IMWG Response Assessment

Qip-Mass Spectrometry in High Risk Smoldering Multiple Myeloma Patients Included in the GEM-CESAR Trial: Comparison with Conventional and Minimal Residual Disease IMWG Response Assessment

Pbp1-Interacting Protein Mkt1 Regulates Virulence and Sexual Reproduction in Cryptococcus neoformans.

The Mkt1–Pbp1 complex promotes mating-type switching by regulating the translation of HO mRNA in Saccharomyces cerevisiae. Here, we performed in vivo immunoprecipitation assays and mass spectrometry analyses in the human fungal pathogen Cryptococcus neoformans to show that Pbp1, a poly(A)-binding protein-binding protein, interacts with Mkt1 containing a PIN like-domain. Association of Pbp1 with Mkt1 was confirmed by co-immunoprecipitation assays. Results of spot dilution growth assays showed that unlike pbp1 deletion mutant strains, mkt1 deletion mutant strains were not resistant to heat stress compared with wild-type. However, similar to the pbp1 deletion mutant strains, the mkt1 deletion mutants exhibited both, defective dikaryotic hyphal production and reduced pheromone gene (MFα1) expression during mating. In addition, deletion of mkt1 caused attenuated virulence in a murine intranasal inhalation model. Taken together, our findings reveal that Mkt1 plays a crucial role in sexual reproduction and virulence in C. neoformans.

Serine-Phosphorylated STAT3 Promotes Tumorigenesis via Modulation of RNA Polymerase Transcriptional Activity.

Deregulated activation of the latent oncogenic transcription factor STAT3 in many human epithelial malignancies, including gastric cancer, has invariably been associated with its canonical tyrosine phosphorylation and enhanced transcriptional activity. By contrast, serine phosphorylation (pS) of STAT3 can augment its nuclear transcriptional activity and promote essential mitochondrial functions, yet the role of pS-STAT3 among epithelial cancers is ill-defined. Here, we reveal that genetic ablation of pS-STAT3 in the gp130 F/F spontaneous gastric cancer mouse model and human gastric cancer cell line xenografts abrogated tumor growth that coincided with reduced proliferative potential of the tumor epithelium. Microarray gene expression profiling demonstrated that the suppressed gastric tumorigenesis in pS-STAT3-deficient gp130 F/F mice associated with reduced transcriptional activity of STAT3-regulated gene networks implicated in cell proliferation and migration, inflammation, and angiogenesis, but not mitochondrial function or metabolism. Notably, the protumorigenic activity of pS-STAT3 aligned with its capacity to primarily augment RNA polymerase II-mediated transcriptional elongation, but not initiation, of STAT3 target genes. Furthermore, by using a combinatorial in vitro and in vivo proteomics approach based on the rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) assay, we identified RuvB-like AAA ATPase 1 (RUVBL1/Pontin) and enhancer of rudimentary homolog (ERH) as interacting partners of pS-STAT3 that are pivotal for its transcriptional activity on STAT3 target genes. Collectively, these findings uncover a hitherto unknown transcriptional role and obligate requirement for pS-STAT3 in gastric cancer that could be extrapolated to other STAT3-driven cancers. SIGNIFICANCE: These findings reveal a new transcriptional role and mandatory requirement for constitutive STAT3 serine phosphorylation in gastric cancer.

Use of Quantitative Immunoprecipitation Mass Spectrometry to Resolve the Complexity of Plasma Cell Populations in Multiple Myeloma

Clonal heterogeneity and evolution of neoplastic plasma cells are common events in multiple myeloma (MM). Myeloma cells can produce M-proteins as intact immunoglobulins (Ig) with or without excess free light chains (FLC), FLC only or, rarely, no detectable M-protein. Ig are characterized by serum immunofixation electrophoresis (IFE) whereas FLC are quantified by turbidimetry (Freelite), two methods that are not always in concordance. Approximately 95% of MM patients have an abnormal FLC ratio but do not always show the abnormal FLC by IFE.

Oxoglutarate dehydrogenase and acetyl-CoA acyltransferase 2 selectively associate with H2A.Z-occupied promoters and are required for histone modifications

Histone H2A.Z plays an essential role in regulating transcriptional rates and memory. Interestingly, H2A.Z-bound nucleosomes are located in both transcriptionally active and inactive promotors, with no clear understanding of the mechanisms via which it differentially regulates transcription. We hypothesized that its functions are mediated through recruitment of regulatory proteins to promoters. Using rapid chromatin immunoprecipitation-mass spectrometry, we uncovered the association of H2A.Z-bound chromatin with the metabolic enzymes, oxoglutarate dehydrogenase (OGDH) and acetyl-CoA acyltransferase 2 (ACAA2). Recombinant green florescence fusion proteins, combined with mutations of predicted nuclear localization signals, confirmed their nuclear localization and chromatin binding. Conclusively, chromatin immunoprecipitation-deep sequencing, confirmed the predominant association of OGDH and ACAA2 with H2A.Z-occupied transcription start sites and enhancers, the former of which we confirmed is conserved in both mouse and human tissue. Furthermore, H2A.Z-deficient human HAP1 cells exhibited reduced chromatin-bound metabolic enzymes, accompanied with reduced posttranslational histone modifications, including acetylation and succinylation. Specifically, knockdown of OGDH diminished H4 succinylation. Thus, the data reveal that select metabolic enzymes are assembled at active, H2A.Z-occupied, promoters, for potential site-directed production of metabolic intermediates that are required for histone modifications.

Immunoprecipitation-mass Spectrometry Reveals New Functions of RCC2 in Breast Cancer Cell Line MDA-MB-468

目的分析RCC2蛋白在乳腺癌MDA-MB-468细胞系中的互作蛋白组并揭示其功能调控网络。 方法运用酶切技术结合一代测序构建携带Flag标签RCC2蛋白过表达载体，慢病毒包装感染构建Flag-RCC2过表达的MDA-MB-468稳转细胞株。采用Flag抗体凝胶珠亲和沉淀，收集蛋白样品进行高敏感度质谱鉴定。生物信息蛋白组学分析互作蛋白组，包括GO注释，KEGG通路富集，构建蛋白互作网络（PPIN）。 结果高可信度164个蛋白聚类分析显示，RCC2及其互作蛋白显著富于核糖体相关通路中，具有RNA与核蛋白体结合能力，揭示其参与蛋白翻译与成熟的过程。 结论成功鉴定RCC2互作蛋白组，揭示RCC2在乳腺癌细胞中发挥新的翻译调控功能。

A simplified and sensitive immunoprecipitation mass spectrometry protocol for the analysis of amyloid-beta peptides in brain tissue

In the field of Alzheimer’s disease, there is an urgent need for novel analytical tools to identify disease-specific biomarkers and to evaluate therapeutics. Preclinical trials commonly employ amyloid beta (Aβ) peptide signatures as a read-out. In this paper, we report a simplified and detailed protocol for robust immunoprecipitation of Aβ in brain tissue prior to mass spectrometric detection exemplified by a study using transgenic mice. The established method employed murine monoclonal and rabbit polyclonal antibodies and was capable of yielding well-reproducible peaks of high intensity with low background signal intensities corresponding to various Aβ forms.

Downregulation of Human DAB2IP Gene Expression in Renal Cell Carcinoma Results in Resistance to Ionizing Radiation.

Renal cell carcinoma (RCC) is known to be highly radioresistant but the mechanisms associated with radioresistance have remained elusive. We found DOC-2/DAB2 interactive protein (DAB2IP) frequently downregulated in RCC, is associated with radioresistance. In this study, we investigated the underlying mechanism regulating radioresistance by DAB2IP and developed appropriate treatment. Several RCC lines with or without DAB2IP expression were irradiated with ionizing radiation (IR) for determining their radiosensitivities based on colony formation assay. To investigate the underlying regulatory mechanism of DAB2IP, immunoprecipitation-mass spectrometry was performed to identify DAB2IP-interactive proteins. PARP-1 expression and enzymatic activity were determined using qRT-PCR, Western blot analysis, and ELISA. In vivo ubiquitination assay was used to test PARP-1 degradation. Furthermore, in vivo mice xenograft model and patient-derived xenograft (PDX) model were used to determine the effect of combination therapy to sensitizing tumors to IR. We notice that DAB2IP-deficient RCC cells acquire IR-resistance. Mechanistically, DAB2IP can form a complex with PARP-1 and E3 ligases that is responsible for degrading PARP-1. Indeed, elevated PARP-1 levels are associated with the IR resistance in RCC cells. Furthermore, PARP-1 inhibitor can enhance the IR response of either RCC xenograft model or PDX model. In this study, we unveil that loss of DAB2IP resulted in elevated PARP-1 protein is associated with IR-resistance in RCC. These results provide a new targeting strategy to improve the efficacy of radiotherapy of RCC.

THE 1946 COHORT: RELATIONSHIPS BETWEEN CEREBRAL AMYLOID PATHOLOGY USING TWO INDEPENDENT METHODS FOR PLASMA AMYLOID BETA MEASUREMENT

There is considerable interest in developing blood-based biomarkers of β-amyloid to screen individuals for the presence of significant brain β-amyloidosis, and so to focus and reduce the requirements for more expensive and/or invasive tests such as amyloid PET or lumbar puncture. Such pre-screening may be particularly valuable in asymptomatic individuals prior to entry into prevention or treatment trials. Emerging techniques for determining blood β-amyloid include immunoassays on the Single Molecule Array (Simoa) platform; and immunoprecipitation mass spectrometry (IP-MS). We compared the Simoa and IP-MS methods as pre-screening blood tests for determining β-amyloid PET positivity based on data from the Insight 46 cohort. These individuals, drawn from the MRC 1946 British Birth Cohort, were all born in mainland Britain in the same week in 1946, followed prospectively, and at age 69–71 all had detailed cognitive testing, blood sampling, 3T-MRI and 18F-Florbetapir-PET. Concentrations of Aβ1-40 and Aβ1-42 in EDTA plasma were determined using commercially available Simoa digital immunoassays (Quanterix: Aβ40 2.0 and Aβ42 2.0; 200 μL each in duplicate) and an IP-MS assay in which stable isotope-labelled recombinant Aβ peptides with known concentrations were added to 250 μL of sample, peptides were extracted using immunoprecipitation, and quantification was performed on a quadrupole-orbitrap high-resolution mass spectrometer. Plasma Aβ1-40 and Aβ1-42 biomarker data will be available for 417 cognitively normal individuals (age: 70.6±0.7yrs), of whom 77 (18.5%) were 18F-Florbetapir β-amyloid-positive (SUVR using eroded white matter reference region cut-off=0.61). Pearson correlation and Bland-Altman analyses will be used to evaluate the agreement between the Simoa and IP-MS techniques. ROC analyses adjusted for age, sex and APOE ε4 carrier status will be used to compare the utility of Aβ1-40, Aβ1-42, and Aβ1-42/40 ratio for both methods in determining amyloid-PET positivity. We will evaluate the absolute and relative reduction in amyloid-PET scan requirements that would result from using each of these methods to pre-screen cognitively normal individuals for amyloid-PET positivity. Using a large population-based cohort of cognitively normal individuals of near identical age with known PET-amyloid status provides a unique opportunity to undertake a direct comparison of two promising blood-based screening methods for quantifying β-amyloid.

Abstract 5170: Targeting FGFR signaling to disrupt cellular cross-talk in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis with a 5 year survival rate of less than 5%. PDAC tumors consist of a desmoplastic stroma, which limits the effectiveness of chemotherapy. Pancreatic stellate cells (PSCs), which form a key part of this stroma, become activated in response to tumor development. Activated PSCs enter a cross-talk with cancer cells to induce tumor cell proliferation and invasion, leading to metastatic spread. Nuclear fibroblast growth factor receptor 1 (nFGFR1) has been found in PSCs at the invasive edge of PDAC tumors. Inhibition of FGFR1 prevents its nuclear translocation in PSCs, which results in decreased invasion in 3D in vitro PDAC models. Nuclear translocation of FGFR1 in PSCs appears to be a vital mechanism that triggers the transcription of key proteins involved in PDAC invasion. We have used a powerful combination of chromatin immunoprecipitation (ChIP-seq) and sub-cellular mass spectrometry to determine the transcriptional targets of nFGFR1 and consequent sub-cellular protein flux. These techniques have allowed us to dissect the functional consequences of FGFR1 knockdown or inhibition in the PSCs. Candidate drivers of invasion are being validated in state-of-the-art 3D in vitro PDAC models. We have extended these functional studies to combination therapy with the clinical agent gemcitabine (targeting cancer cells) and all-trans retinoic acid (ATRA, modulating PSCs), providing translational relevance for our findings. We are embarking on validating this novel strategy using in vivo co-culture xenograft models with specific reference to FGFR1. Effectively disrupting the cross-talk between the tumor and stroma, either alone or in combination with other therapies, could translate to improved therapeutic responses in PDAC patients in the clinic. Citation Format: Abigail Coetzee, Edward Carter, James Heward, Faraz Mardakheh, Richard Grose, Hemant Kocher. Targeting FGFR signaling to disrupt cellular cross-talk in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5170.

The FUS-DDIT3 Interactome in Myxoid Liposarcoma

Myxoid liposarcoma is a malignant lipogenic tumor that develops in deep soft tissues. While local control rates are good, current chemotherapy options remain ineffective against metastatic disease. Myxoid liposarcoma is characterized by the FUS-DDIT3 fusion oncoprotein that is proposed to function as an aberrant transcription factor, but its exact mechanism of action has remained unclear. To identify the key functional interacting partners of FUS-DDIT3, this study utilized immunoprecipitation-mass spectrometry (IP-MS) to identify the FUS-DDIT3 interactome in whole cell lysates of myxoid liposarcoma cells, and results showed an enrichment of RNA processing proteins. Further quantitative MS analyses of FUS-DDIT3 complexes isolated from nuclear lysates showed that members of several chromatin regulatory complexes were present in the FUS-DDIT3 interactome, including NuRD, SWI/SNF, PRC1, PRC2, and MLL1 COMPASS-like complexes. Co-immunoprecipitation validated the associations of FUS-DDIT3 with BRG1/SMARCA4, BAF155/SMARCC1, BAF57/SMARCE1, and KDM1A. Data from this study provides candidates for functional validation as potential therapeutic targets, particularly for emerging epigenetic drugs.

Proteomics Approaches for Biomarker and Drug Target Discovery in ALS and FTD.

Neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are increasing in prevalence but lack targeted therapeutics. Although the pathological mechanisms behind these diseases remain unclear, both ALS and FTD are characterized pathologically by aberrant protein aggregation and inclusion formation within neurons, which correlates with neurodegeneration. Notably, aggregation of several key proteins, including TAR DNA binding protein of 43 kDa (TDP-43), superoxide dismutase 1 (SOD1), and tau, have been implicated in these diseases. Proteomics methods are being increasingly applied to better understand disease-related mechanisms and to identify biomarkers of disease, using model systems as well as human samples. Proteomics-based approaches offer unbiased, high-throughput, and quantitative results with numerous applications for investigating proteins of interest. Here, we review recent advances in the understanding of ALS and FTD pathophysiology obtained using proteomics approaches, and we assess technical and experimental limitations. We compare findings from various mass spectrometry (MS) approaches including quantitative proteomics methods such as stable isotope labeling by amino acids in cell culture (SILAC) and tandem mass tagging (TMT) to approaches such as label-free quantitation (LFQ) and sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) in studies of ALS and FTD. Similarly, we describe disease-related protein-protein interaction (PPI) studies using approaches including immunoprecipitation mass spectrometry (IP-MS) and proximity-dependent biotin identification (BioID) and discuss future application of new techniques including proximity-dependent ascorbic acid peroxidase labeling (APEX), and biotinylation by antibody recognition (BAR). Furthermore, we explore the use of MS to detect post-translational modifications (PTMs), such as ubiquitination and phosphorylation, of disease-relevant proteins in ALS and FTD. We also discuss upstream technologies that enable enrichment of proteins of interest, highlighting the contributions of new techniques to isolate disease-relevant protein inclusions including flow cytometric analysis of inclusions and trafficking (FloIT). These recently developed approaches, as well as related advances yet to be applied to studies of these neurodegenerative diseases, offer numerous opportunities for discovery of potential therapeutic targets and biomarkers for ALS and FTD.

Systematic assessment of antibody selectivity in plasma based on a resource of enrichment profiles

There is a strong need for procedures that enable context and application dependent validation of antibodies. Here, we applied a magnetic bead assisted workflow and immunoprecipitation mass spectrometry (IP-MS/MS) to assess antibody selectivity for the detection of proteins in human plasma. A resource was built on 414 IP experiments using 157 antibodies (targeting 120 unique proteins) in assays with heat-treated or untreated EDTA plasma. For each protein we determined their antibody related degrees of enrichment using z-scores and their frequencies of identification across all IP assays. Out of 1,313 unique endogenous proteins, 426 proteins (33%) were detected in >20% of IPs, and these background components were mainly comprised of proteins from the complement system. For 45% (70/157) of the tested antibodies, the expected target proteins were enriched (z-score ≥ 3). Among these 70 antibodies, 59 (84%) co-enriched other proteins beside the intended target and mainly due to sequence homology or protein abundance. We also detected protein interactions in plasma, and for IGFBP2 confirmed these using several antibodies and sandwich immunoassays. The protein enrichment data with plasma provide a very useful and yet lacking resource for the assessment of antibody selectivity. Our insights will contribute to a more informed use of affinity reagents for plasma proteomics assays.

PF199 TARGETING BRD4 IN MLL‐PTD AML

Background:Mixed lineage leukemia (MLL) gene encodes for a nuclear protein that regulates genes involved in development and hematopoiesis. De‐regulation of MLL by chromosome translocations causes acute myeloid leukemia (AML). In addition, about 5–11% of AML patients have MLL‐partial tandem duplication (PTD) mutations. MLL‐PTD is associated with poor prognosis and shorter remission duration in AML. Preliminary studies from our group indicate that when MLL‐PTD is knockdown in AML blasts we observe a mRNA expression profile that is very similar to that of bromodomain 4 (BRD4) inhibition in AML.Aims:We hypothesized that MLL‐PTD interacts with BRD4 forming a complex that regulate key genes in MLL‐PTD positive AML. Therefore treatment with BRD4 inhibitors, such as JQ1, could be a valid therapeutic strategy for MLL‐PTD AML patients.Methods:To identify proteins forming the transcriptional complex regulated by BRD4 and MLL, we conducted a Rapid Immunoprecipitation Mass spectrometry of Endogenous protein (RIME) study using BRD4 and MLL antibodies. Successively, we treated MLL‐PTD positive primary patient cells and CD34+ cord blood from healthy donors as controls with JQ1, and conducted RNA‐seq and ChIP‐seq studies. Finally, we conducted in vitro and in vivo studies on MLL‐PTD harboring samples to assess JQ1 treatment efficiency in inducing cell death in MLL‐PTD AML.Results:RIME analysis using BRD4 and MLL antibodies showed that the 75% of the proteins were in common between the two groups. PANTHER gene ontology analysis identified common pathways between them, suggesting that MLL and BRD4 could be part of similar protein complexes. Next, we treated MLL‐PTD+ primary patient cells, and CD34+ cord blood cells as controls with JQ1 or vehicle, and conducted RNA‐seq and ChIP‐seq studies. RNA expression and peak intensity on promoter region results were combined to identify genes aberrantly expressed in MLL‐PTD+ samples. We identified 98 up‐regulated and 32 down‐regulated genes that were differentially expressed d by JQ1 treatment only in AML samples. The top deregulated genes were validated by RT‐qPCR and ChIP in both human and murine MLL‐PTD+ samples. Next, we treated the MLL‐PTD+ AML cell line (EOL1) and primary murine AML cells from Mll‐PTD/Flt3‐ITD model with JQ1 and showed that JQ1 treatment induced more apoptosis and reduction of living cells in MLL‐PTD+ models respect the relative controls (fold change living cells at 48 hours WT: 0.81, Mll‐PTD+: 0.42, p‐value: 0.0247). Last, treatment of the Mll‐PTD/Flt3‐ITD xenotranplanted BoyJ mice with JQ1 significantly increased their survival compared with controls (median survival CTRL: 75 days, JQ1: 109 days, Mantel‐Cox p‐value: 0.0195). In addition, we observed a reduced engraftment in serial re‐transplantation assays.Summary/Conclusion:Our data support developing a therapeutic strategy to target BRD4 in MLL‐PTD AML.image

Circulating Levels of Interferon Regulatory Factor-5 Associates With Subgroups of Systemic Lupus Erythematosus Patients

Systemic Lupus Erythematosus (SLE) is a heterogeneous autoimmune disease, which currently lacks specific diagnostic biomarkers. The diversity within the patients obstructs clinical trials but may also reflect differences in underlying pathogenesis. Our objective was to obtain protein profiles to identify potential general biomarkers of SLE and to determine molecular subgroups within SLE for patient stratification. Plasma samples from a cross-sectional study of well-characterized SLE patients (n = 379) and matched population controls (n = 316) were analyzed by antibody suspension bead array targeting 281 proteins. To investigate the differences between SLE and controls, Mann–Whitney U-test with Bonferroni correction, generalized linear modeling and receiver operating characteristics (ROC) analysis were performed. K-means clustering was used to identify molecular SLE subgroups. We identified Interferon regulating factor 5 (IRF5), solute carrier family 22 member 2 (SLC22A2) and S100 calcium binding protein A12 (S100A12) as the three proteins with the largest fold change between SLE patients and controls (SLE/Control = 1.4, 1.4, and 1.2 respectively). The lowest p-values comparing SLE patients and controls were obtained for S100A12, Matrix metalloproteinase-1 (MMP1) and SLC22A2 (padjusted = 3 × 10−9, 3 × 10−6, and 5 × 10−6 respectively). In a set of 15 potential biomarkers differentiating SLE patients and controls, two of the proteins were transcription factors, i.e., IRF5 and SAM pointed domain containing ETS transcription factor (SPDEF). IRF5 was up-regulated while SPDEF was found to be down-regulated in SLE patients. Unsupervised clustering of all investigated proteins identified three molecular subgroups among SLE patients, characterized by (1) high levels of rheumatoid factor-IgM, (2) low IRF5, and (3) high IRF5. IRF5 expressing microparticles were analyzed by flow cytometry in a subset of patients to confirm the presence of IRF5 in plasma and detection of extracellular IRF5 was further confirmed by immunoprecipitation-mass spectrometry (IP-MS). Interestingly IRF5, a known genetic risk factor for SLE, was detected extracellularly and suggested by unsupervised clustering analysis to differentiate between SLE subgroups. Our results imply a set of circulating molecules as markers of possible pathogenic importance in SLE. We believe that these findings could be of relevance for understanding the pathogenesis and diversity of SLE, as well as for selection of patients in clinical trials.

Identification of low level monoclonality using quantitative immunoprecipitation mass spectrometry (QIP-MS) as a first line monoclonal gammopathy screening strategy

The venous thromboembolism, which may be caused by increased platelet activation, is a risk factor for tumor prognosis. We determined the platelet activation status for diagnosis and predicting postoperative prognosis of hepatocellular carcinoma.We conducted a prospective study of 191 patients diagnosed with HCC at Zhongshan Hospital from April 2016 to July 2016 as well as 99 healthy people. The platelet activation status was assessed by 2 platelet markers, PAC-1 and CD62p, using flow cytometry. The patients were treated with TACE or resection and monitored for ≥6 months. The diagnostic value of marker-positive platelets was determined by the receiver operating characteristic curve and the postoperative value were analyzed using the Kaplan-Meier method and COX regression model.All the 3 groups with high levels of marker-positive platelets were likely to be diagnosed with HCC and the PAC-1+ percentage had the best efficacy. The univariate analysis showed that the levels of PAC-1+ and CD62p+ platelets was risker factors for poor postoperative prognosis after both TACE and resection. Moreover, the multivariate analysis revealed that the level of PAC-1+ platelets was an independent risk factor for poor prognosis.The PAC-1+ percentage of platelets is a new indicator for diagnosis and predicting postoperative prognosis.

MON-483 FOXO1 Contributes to Somatotrope Differentiation through Regulation of Key Transcription Factors

Pituitary gland function is regulated by the activity of various transcription factors which control cell fate decisions leading to cellular differentiation and hormone production. FOXO1 is necessary for the proper timing of somatotrope differentiation and for somatotrope function, but the exact mechanism of action has yet to be elucidated. Recent data implicate FOXO1 in the regulation of genes important for somatotrope differentiation including Gh1, Neurod4, and Pou1f1. Previously, a mouse model with conditional deletion of Foxo1 from the developing pituitary gland displayed reduced Gh1 and Neurod4 transcripts as early as embryonic day 18.5. Additional data from adult animals with conditional deletion of both Foxo1 and Foxo3 from the pituitary gland have a similar reduction in Neurod4 and Gh1, as well as Pou1f1. In order to investigate the mechanism by which FOXO1 regulates pituitary gland gene expression and confirm in vivo findings, the somatotrope-like cell line MtT/S was treated with FOXO1 inhibitor, AS184256, for 24 hours at various concentrations. Neurod4 and Pou1f1 were the most severely affected genes with a dose-dependent reduction in transcript at inhibitor concentrations as low as 30 nM (p < 0.05). Gh1 transcripts were significantly reduced at 3 μM (p < 0.01). To ascertain whether FOXO1 binds to regulatory regions of key somatotrope genes, ChIP-seq was performed using the MtT/S cell line and anti-FOXO1 antibody. This study identified FOXO1 interaction with genomic regions associated with Neurod4, Pou1f1, and Gh1. Interestingly, the FOXO1 binding intervals associated with Neurod4 and Pou1f1 are highly conserved between rat and human, indicating these sequences are important for survival. These data strongly suggest FOXO1 directly contributes to the transcriptional control of genes important for somatotrope differentiation. Investigation is also underway to identify proteins within the FOXO1 transcriptional complex. Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) was performed using the MtT/S cell line for this purpose. Proteins important for signal transduction and transcriptional regulation, including PKC, LRPPRC, and YBX1, were enriched in chromatin containing FOXO1, suggesting that they contribute to transcriptional control of FOXO1 targets. Overall, the emerging mechanism for FOXO1 control of somatotrope differentiation likely involves direct regulation of Neurod4, Pou1f1, and Gh1. These novel findings contribute to the much-needed understanding of pituitary cell fate decisions.