RESUMEN
Mutations or aberrant upregulation of EZH2 occur frequently in human cancers, yet clinical benefits of EZH2 inhibitor (EZH2i) remain unsatisfactory and limited to certain hematological malignancies. We profile global posttranslational histone modification changes across a large panel of cancer cell lines with various sensitivities to EZH2i. We report here oncogenic transcriptional reprogramming mediated by MLL1's interaction with the p300/CBP complex, which directs H3K27me loss to reciprocal H3K27ac gain and restricts EZH2i response. Concurrent inhibition of H3K27me and H3K27ac results in transcriptional repression and MAPK pathway dependency in cancer subsets. In preclinical models encompassing a broad spectrum of EZH2-aberrant solid tumors, a combination of EZH2 and BRD4 inhibitors, or a triple-combination including MAPK inhibition display robust efficacy with very tolerable toxicity. Our results suggest an attractive precision treatment strategy for EZH2-aberrant tumors on the basis of tumor-intrinsic MLL1 expression and concurrent inhibition of epigenetic crosstalk and feedback MAPK activation.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Proteína Potenciadora del Homólogo Zeste 2/genética , N-Metiltransferasa de Histona-Lisina/fisiología , Proteína de la Leucemia Mieloide-Linfoide/fisiología , Animales , Carcinogénesis/genética , Proteínas de Ciclo Celular , Línea Celular Tumoral , Epigénesis Genética/genética , Epigenómica/métodos , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Código de Histonas/efectos de los fármacos , Código de Histonas/genética , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Histonas/fisiología , Humanos , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Mutación , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiología , Complejo Represivo Polycomb 2/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Activación Transcripcional , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Factores de Transcripción p300-CBP/fisiologíaRESUMEN
In a previous work we demonstrated that CHO protease caused fragmentation of an expressed bispecific antibody (bsAb) and this detrimental host cell protein (HCP) can be effectively removed through an optimized Protein A wash step. In addition, preliminary evidence suggested that the responsible protease belongs to the threonine or cysteine protease family. In the current study, this protease was further identified as cathepsin B. First, we identified several CHO proteases in the further fractionated Protein A wash using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and this allowed us to select four candidate proteases. Next, by examining the cleavage pattern of each individual protease and comparing it with that observed during purification, cathepsin B was identified as the protease responsible for the observed bsAb fragmentation.
Asunto(s)
Anticuerpos Biespecíficos , Péptido Hidrolasas , Animales , Anticuerpos Biespecíficos/genética , Células CHO , Catepsina B/genética , Cromatografía Liquida , Cricetinae , Cricetulus , Péptido Hidrolasas/metabolismo , Proteína Estafilocócica A , Espectrometría de Masas en TándemRESUMEN
B-cell lymphomas are heterogeneous blood disorders with limited therapeutic options, largely because of their propensity to relapse and become refractory to treatments. Carabin, a key suppressor of B-cell receptor signaling and proliferation, is inactivated in B-cell lymphoma by unknown mechanisms. Here, we identify prolyl 4-hydroxylase 2 (P4HA2) as a specific proline hydroxylase of Carabin. Carabin hydroxylation leads to its proteasomal degradation, thereby activating the Ras/extracellular signal-regulated kinase pathway and increasing B-cell lymphoma proliferation. P4HA2 is undetectable in normal B cells but upregulated in the diffuse large B-cell lymphoma (DLBCL), driving Carabin inactivation and lymphoma proliferation. Our results indicate that P4HA2 is a potential prognosis marker for DLBCL and a promising pharmacological target for developing treatment of molecularly stratified B-cell lymphomas.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Biomarcadores de Tumor/metabolismo , Proliferación Celular , Linfoma de Células B Grandes Difuso/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas de Neoplasias/metabolismo , Prolil Hidroxilasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Proteínas Activadoras de GTPasa , Humanos , Hidroxilación , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Proteínas de Neoplasias/genética , Prolil Hidroxilasas/genética , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , ProteolisisRESUMEN
Epithelial barrier disruption is a major cause of inflammatory bowel disease (IBD); however, the mechanism through which epigenetic regulation modulates intestinal epithelial integrity remains largely undefined. Here we show that EZH2, the catalytic subunit of polycomb repressive complex (PRC2), is indispensable for maintaining epithelial cell barrier integrity and homeostasis under inflammatory conditions. In accordance with reduced EZH2 expression in patients, the inactivation of EZH2 in IECs sensitizes mice to DSS- and TNBS-induced experimental colitis. Conversely, EZH2 overexpression in the intestinal epithelium renders mice more resistant to colitis. Mechanistically, the genes encoding TRAF2/5 are held in a finely tuned bivalent status under inflammatory conditions. EZH2 deficiency potentiates the expression of these genes to enhance TNFα-induced NF-κB signaling, thereby leading to uncontrolled inflammation. More importantly, we show that EZH2 depletion compromises the protective role of NF-κB signaling in cell survival by directly up-regulating ITCH, a well-known E3 ligase that degrades the c-FLIP protein. Thus, our findings highlight an epigenetic mechanism by which EZH2 integrates the multifaceted effects of TNFα signaling to promote the inflammatory response and apoptosis in colitis.
Asunto(s)
Apoptosis , Colitis/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Epigénesis Genética , Mucosa Intestinal/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Colitis/inducido químicamente , Colitis/genética , Colitis/patología , Sulfato de Dextran/toxicidad , Proteína Potenciadora del Homólogo Zeste 2/genética , Humanos , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Mucosa Intestinal/patología , Ratones , Ratones Noqueados , FN-kappa B/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factor 2 Asociado a Receptor de TNF/genética , Factor 2 Asociado a Receptor de TNF/metabolismo , Factor 5 Asociado a Receptor de TNF/genética , Factor 5 Asociado a Receptor de TNF/metabolismo , Factor de Necrosis Tumoral alfa/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Ultrafiltration/diafiltration (UF/DF) is typically the final step in downstream processing of recombinant monoclonal antibody (mAb) products, which serves for protein concentration and buffer exchange. For UF/DF membranes composed of regenerated cellulose (RC), sanitization with 0.1 M sodium hydroxide is generally recommended by the supplier, but it may not be sufficient for reducing bioburden during large scale manufacturing. Therefore, more stringent sanitization methods for RC membranes are required. However, chemicals used in such sanitization step may disrupt membrane integrity, while the corresponding residuals may reduce product quality. Previous work has shown that high concentration of sodium hydroxide or addition of peracetic acid (PAA) can effectively reduce bioburden, but their effects on the RC membranes remain unknown. In this work, we assessed the impact of two sanitization methods, 0.5 M sodium hydroxide and 30 mM PAA in combination with 0.5 M sodium hydroxide, on membrane integrity and protein quality of Millipore and pall corporation (PALL) membranes. Both methods showed a similar impact as the control after performing 15 cycles. However, the addition of PAA may cause residual chemical concerns, therefore, 0.5 M sodium hydroxide was recommended as an effective and safe sanitization method for RC UF/DF membranes.
Asunto(s)
Celulosa , Ultrafiltración , Ultrafiltración/métodos , Hidróxido de Sodio , Proteínas , Membranas ArtificialesRESUMEN
AMP-activated protein kinase (AMPK) is a conserved energy sensor that plays roles in diverse biological processes via phosphorylating various substrates. Emerging studies have demonstrated the regulatory roles of AMPK in DNA repair, but the underlying mechanisms remain to be fully understood. Herein, using mass spectrometry-based proteomic technologies, we systematically investigate the regulatory network of AMPK in DNA damage response (DDR). Our system-wide phosphoproteome study uncovers a variety of newly-identified potential substrates involved in diverse biological processes, whereas our system-wide histone modification analysis reveals a link between AMPK and histone acetylation. Together with these findings, we discover that AMPK promotes apoptosis by phosphorylating apoptosis-stimulating of p53 protein 2 (ASPP2) in an irradiation (IR)-dependent manner and regulates histone acetylation by phosphorylating histone deacetylase 9 (HDAC9) in an IR-independent manner. Besides, we reveal that disrupting the histone acetylation by the bromodomain BRD4 inhibitor JQ-1 enhances the sensitivity of AMPK-deficient cells to IR. Therefore, our study has provided a resource to investigate the interplay between phosphorylation and histone acetylation underlying the regulatory network of AMPK, which could be beneficial to understand the exact role of AMPK in DDR.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Histonas , Proteínas Quinasas Activadas por AMP/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Acetilación , Proteómica , Factores de Transcripción/metabolismo , Fosforilación/fisiología , Daño del ADNRESUMEN
SKP1-CUL1-F-box (SCF) ubiquitin ligases play fundamental roles in cellular functions. Typically, substrate phosphorylation is required for SCF recognition and subsequent degradation. However, phospho-dependent substrates remain largely unidentified. Here, using quantitative phoshoproteome approach, we performed a system-wide investigation of phospho-dependent SCF substrates. This strategy identified diverse phospho-dependent candidates. Biochemical verification revealed a mechanism by which SCFFBXO22 recognizes the motif XXPpSPXPXX as a conserved phosphodegron to target substrates for destruction. We further demonstrated BAG3, a HSP70 co-chaperone, is a bona fide substrate of SCFFBXO22. FBXO22 mediates BAG3 ubiquitination and degradation that requires ERK-dependent BAG3 phosphorylation at S377. FBXO22 depletion or expression of a stable BAG3 S377A mutant promotes tumor growth via defects in apoptosis and cell cycle progression in vitro and in vivo. In conclusion, our study identified broad phosphorylation-dependent SCF substrates and demonstrated a phosphodegron recognized by FBXO22 and a novel ERK-FBXO22-BAG3 axis involved in tumorigenesis.
Asunto(s)
Carcinogénesis , Proteínas F-Box , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Transformación Celular Neoplásica , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Humanos , Fosforilación , Receptores Citoplasmáticos y Nucleares/metabolismo , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
The CRISPR/Cas9 system, originally derived from the prokaryotic adaptive immune system, has been developed as efficient genome editing tools. It enables precise gene manipulation on chromosomal DNA through the specific binding of programmable sgRNA to target DNA, and the Cas9 protein, which has endonuclease activity, will cut a double strand break at specific locus. However, Cas9 is a foreign protein in mammalian cells, and the potential risks associated with its introduction into mammalian cells are not fully understood. In this study, we performed pull-down and mass spectrometry (MS) analysis of Streptococcus pyogenes Cas9 (SpyCas9) interacting proteins in HEK293T cells and showed that the majority of Cas9-associated proteins identified by MS were localized in the nucleolus. Interestingly, we further discovered that the Cas9 protein contains a sequence encoding a nucleolus detention signal (NoDS). Compared with wild-type (WT) Cas9, NoDS-mutated variants of Cas9 (mCas9) are less stable, although their gene editing activity is minimally affected. Overexpression of WT Cas9, but not mCas9, causes general effects on transcription and protein translation in the host cell. Overall, identification of NoDS in Cas9 will improve the understanding of Cas9's biological function in vivo, and the removal of NoDS in Cas9 may enhance its safety for future clinical use.
RESUMEN
The selection of a data processing method for use in mass spectrometry-based label-free proteome quantification contributes significantly to its accuracy and precision. In this study, we comprehensively evaluated 7 commonly-used label-free quantification methods (MaxQuant-Spectrum count, MaxQuant-iBAQ, MaxQuant-LFQ, MaxQuant-LFAQ, Proteome Discoverer, MetaMorpheus, TPP-StPeter) with a focus on missing values, precision, accuracy, selectivity, and reproducibility of low abundance protein quantification in both single shot and fractionation. Our results showed that among the tested strategies, MaxQuant in MaxLFQ mode outperformed other strategies in terms of accuracy and precision in both whole proteome and low abundance proteome quantification, whereas the Proteome Discoverer (PD) strategy using SEQUEST as a search engine performed better in terms of quantifiable low abundance proteome coverage. We subsequently applied the PD and MaxLFQ strategies in a blood proteomic dataset and found that many FDA-approved tumor prognostic biomarkers could be identified as well as quantified using the PD strategy, indicating the potential advantage of PD in label-free quantification studies. These results provide a reference for method choice in label-free quantification data analysis. SIGNIFICANCE: Mass spectrometry-based label-free quantification methods play an important role in label-free proteome data analysis. In this study, we evaluated 7 commonly-used label-free quantification methods with respect to the following aspects: missing values, precision, accuracy, selectivity, and reproducibility for low abundance protein quantification. The results showed that, among the strategies evaluated, the PD strategy with SEQUEST as a search engine performed better in terms of low abundance protein coverage. This study provides a reference for method choice in label-free quantification data analysis.