Anomalous Constitutive Src Kinase Activity Promotes B Lymphoma Survival and Growth

Background: Previously we have shown that B cell receptor (BCR) expression and B cell receptor signaling pathways are important for the basal growth of B lymphoma cells. In particular we have shown that the activation of Syk, a non-src family protein tyrosine kinase and the mitogen activated protein kinases (MAPK), ERK and JNK that mediate BCR signals are required for the constitutive growth of B lymphoma cells. Since src family protein tyrosine kinases (SFKs) like Lyn are known to be needed for the phosphorylation of BCR co-receptors, Ig-alpha and Ig-beta, we hypothesized that one or more SFKs will be constitutively activated in B lymphoma cells and may be necessary for B lymphoma growth. Results: Src kinase activity was found to be constitutively high in many murine and human B lymphoma cell lines and primary lymphoma samples. The specific pharmacological inhibitors of SFKs, PP1 and PP2 inhibited the proliferation of a number of both murine and human B lymphomas in a dose-dependent manner. Importantly, dasatinib (BMS-354825), an oral dual BCR-ABL and SFK specific inhibitor inhibited the growth of B lymphomas in the nanomolar range in vitro and strongly inhibited a mouse lymphoma growth in vivo. Among the SFKs, Lyn is predominantly phosphorylated and Lyn-specific small interfering RNA inhibited the growth of B lymphomas, supporting an important role for Lyn in B lymphoma growth. Suppression of SFK activity blocks BCR mediated signaling pathways. PMA or CpG can partially reverse the growth inhibition induced by SFK inhibition. Although blocking SFK activity inhibited the growth of a number of B lymphomas, some lymphomas such as SudHL-4, SudHL-6, OCI-Ly3 and OCI-Ly10 are more resistant due to an increased expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL. Conclusions: These studies further support our concept that BCR signaling pathways are important for the continued growth of established B lymphoma cells. Some of the intermediates in this BCR pathway are potential immunotherapeutic targets. In particular, inhibition of SFK activity alone or in synergy with inhibition of the prosurvival Bcl-2 proteins holds promise in developing more effective treatments for B lymphoma patients

Scalable Incomplete Multi-View Clustering with Structure Alignment

The success of existing multi-view clustering (MVC) relies on the assumption that all views are complete. However, samples are usually partially available due to data corruption or sensor malfunction, which raises the research of incomplete multi-view clustering (IMVC). Although several anchor-based IMVC methods have been proposed to process the large-scale incomplete data, they still suffer from the following drawbacks: i) Most existing approaches neglect the inter-view discrepancy and enforce cross-view representation to be consistent, which would corrupt the representation capability of the model; ii) Due to the samples disparity between different views, the learned anchor might be misaligned, which we referred as the Anchor-Unaligned Problem for Incomplete data (AUP-ID). Such the AUP-ID would cause inaccurate graph fusion and degrades clustering performance. To tackle these issues, we propose a novel incomplete anchor graph learning framework termed Scalable Incomplete Multi-View Clustering with Structure Alignment (SIMVC-SA). Specially, we construct the view-specific anchor graph to capture the complementary information from different views. In order to solve the AUP-ID, we propose a novel structure alignment module to refine the cross-view anchor correspondence. Meanwhile, the anchor graph construction and alignment are jointly optimized in our unified framework to enhance clustering quality. Through anchor graph construction instead of full graphs, the time and space complexity of the proposed SIMVC-SA is proven to be linearly correlated with the number of samples. Extensive experiments on seven incomplete benchmark datasets demonstrate the effectiveness and efficiency of our proposed method. Our code is publicly available at https://github.com/wy1019/SIMVC-SA

The Role of MAPKs in B Cell Receptor-Induced Down-Regulation of Egr-1 in Immature B Lymphoma Cells

Cross-linking of the B cell receptor (BCR) on the immature B lymphoma cell line BKS-2 induces growth inhibition and apoptosis accompanied by rapid down-regulation of the immediate-early gene egr-1. In these lymphoma cells, egr-1 is expressed constitutively and has a prosurvival role, as Egr-1-specific antisense oligonucleotides or expression of a dominant-negative inhibitor of Egr-1 also prevented the growth of BKS-2 cells. Moreover, enhancement of Egr-1 protein with phorbol 12-myristate 13-acetate or an egr-1 expression vector rescued BKS-2 cells from BCR signal-induced growth inhibition. Nuclear run-on and mRNA stability assays indicated that BCR-derived signals act at the transcriptional level to reduce egr-1 expression. Inhibitors of ERK and JNK (but not of p38 MAPK) reduced egr-1 expression at the protein level. Transcriptional regulation appears to have a role because egr-1 promoter-driven luciferase expression was reduced by ERK and JNK inhibitors. Promoter truncation experiments suggested that several serum response elements are required for MAPK-mediated egr-1 expression. Our study suggests that BCR signals reduce egr-1 expression by inhibiting activation of ERK and JNK. Unlike ERK and JNK, p38 MAPK reduces constitutive expression of egr-1. Unlike the immature B lymphoma cells, normal immature B cells did not exhibit constitutive MAPK activation. BCR-induced MAPK activation was modest and transient with a small increase in egr-1 expression in normal immature B cells consistent with their inability to proliferate in response to BCR cross-linking

Identification and characterization of circular RNAs in mammary gland tissue from sheep at peak lactation and during the nonlactating period

Circular RNAs are a class of noncoding RNA with a widespread occurrence in eukaryote tissues, and with some having been demonstrated to have clear biological function. In sheep, little is known about the role of circular RNAs in mammary gland tissue, and therefore an RNA sequencing approach was used to compare mammary gland tissue expression of circular RNAs in 9 Small Tail Han sheep at peak lactation, and subsequently when they were not lactating. These 9 sheep had their RNA pooled for analysis into 3 libraries from peak lactation and 3 from the nonlactating period. A total of 3,278 and 1,756 circular RNAs were identified in the peak lactation and nonlactating mammary gland tissues, respectively, and the expression and identity of 9 of them was confirmed using reverse transcriptase-polymerase chain reaction analysis and DNA sequencing. The type, chromosomal location and length of the circular RNAs identified were ascertained. Forty upregulated and one downregulated circular RNAs were characterized in the mammary gland tissue at peak lactation compared with the nonlactating mammary gland tissue. Gene ontology enrichment analysis revealed that the parental genes of these differentially expressed circular RNAs were related to molecular function, binding, protein binding, ATP binding, and ion binding. Five differentially expression circular RNAs were selected for further analysis to predict their target microRNAs, and some microRNAs reportedly associated with the development of the mammary gland were found in the constructed circular RNA–microRNA network. This study reveals the expression profiles and characterization of circular RNAs at 2 key stages of mammary gland activity, thereby providing an improved understanding of the roles of circular RNAs in the mammary gland of sheep

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology

Modulation of β-Catenin Signaling by Glucagon Receptor Activation

The glucagon receptor (GCGR) is a member of the class B G protein–coupled receptor family. Activation of GCGR by glucagon leads to increased glucose production by the liver. Thus, glucagon is a key component of glucose homeostasis by counteracting the effect of insulin. In this report, we found that in addition to activation of the classic cAMP/protein kinase A (PKA) pathway, activation of GCGR also induced β-catenin stabilization and activated β-catenin–mediated transcription. Activation of β-catenin signaling was PKA-dependent, consistent with previous reports on the parathyroid hormone receptor type 1 (PTH1R) and glucagon-like peptide 1 (GLP-1R) receptors. Since low-density-lipoprotein receptor–related protein 5 (Lrp5) is an essential co-receptor required for Wnt protein mediated β-catenin signaling, we examined the role of Lrp5 in glucagon-induced β-catenin signaling. Cotransfection with Lrp5 enhanced the glucagon-induced β-catenin stabilization and TCF promoter–mediated transcription. Inhibiting Lrp5/6 function using Dickkopf-1(DKK1) or by expression of the Lrp5 extracellular domain blocked glucagon-induced β-catenin signaling. Furthermore, we showed that Lrp5 physically interacted with GCGR by immunoprecipitation and bioluminescence resonance energy transfer assays. Together, these results reveal an unexpected crosstalk between glucagon and β-catenin signaling, and may help to explain the metabolic phenotypes of Lrp5/6 mutations

Crystal structures and mechanisms of two enzymes involved in the degradation of tyrosine and biphenyl

Homogentisate 1,2-dioxygenase (HGO), the third enzyme in tyrosine degradation, is an extradiol-type dioxygenase that utilizes nonheme Fe(II) and dioxygen to catalyze the ring cleavage of homogentisate (HGA). Its chemical mechanism may be studied by structural and biochemical approaches. Crystal structures of anaerobically prepared human HGO and the HGO:HGA complex have been determined to 1.6 and 1.8 Å resolution. The structure of the HGO:HGA complex shows that the substrate binds at the active site in a mode that differs from that predicted by a prior modeling study. Based on the structure, a revised mechanism is proposed. HGO is a potential target of inhibition to treat hepatorenal tyrosinemia type 1 (HT1). Biochemical data indicated that 3-Cl HGA is an effective inhibitor of human HGO and the structure of HGO:3-Cl HGA complex has been determined to 1.7 Å resolution to understand the inhibition. 3-Cl HGA binds differently than the substrate, which suggests that the slow catalysis may be due to the predominant non-productive binding mode in the crystal structure. The activity of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) hydrolase (BphD), a C-C bond hydrolase, is a key determinant in the aerobic transformation of polychlorinated biphenyls (PCBs). The failure of the bph pathway to efficiently process PCBs and PCB metabolites limits strategies for bioremediation. We study mechanisms of BphD catalysis and catalytic failure using structural and biochemical approaches. Crystal structures of BphD LB400 (BphD from Burkholderia strain LB400), its S112C mutant, the S112C:HOPDA complex, and S112A mutant have been determined at 1.6 Å resolution. BphDLB400 is a tetramer. Each monomer has a typical α/β hydrolase fold and is divisible into core and lid domains. The active site is located between the two domains and consists of polar and non-polar parts including a catalytic triad, S112-H265-D237. The mechanism of C-C bond hydrolases is a subject of debate. Recent biochemical evidence favors a mechanism that generates a gem-diol intermediate following base-catalyzed attack by water. In the S112C:HOPDA structure, C112 covalently binds HOPDA at the C6 position, forming a complex that resembles a tetrahedral intermediate consistent with a protein-nucleophile mechanism

Seven Fatty Acid Metabolism-Related Genes as Potential Biomarkers for Predicting the Prognosis and Immunotherapy Responses in Patients with Esophageal Cancer

Background: Esophageal cancer (ESCA) is a major cause of cancer-related mortality worldwide. Altered fatty acid metabolism is a hallmark of cancer. However, studies on the roles of fatty acid metabolism-related genes (FRGs) in ESCA remain limited. Method: We identified differentially expressed FRGs (DE-FRGs). Then, the DE-FRGs prognostic model was constructed and validated using a comprehensive analysis. Moreover, the correlation between the risk model and clinical characteristics was investigated. A nomogram for predicting survival was established and evaluated. Subsequently, the difference in tumor microenvironment (TME) was compared between two risk groups. The sensitivity of key DE-FRGs to chemotherapeutic interventions and their correlation with immune cells were investigated. Finally, DEGs between two risk groups were measured and the prognostic value of key DE-FRGs in ESCA was confirmed in other databases. Results: A prognostic model was constructed based on seven selected DEG-FRGs. TNM staging and CD8+ T cells were significantly correlated with high-risk groups. Low-risk groups exhibited more infiltrated M0 macrophages, an activation of type II interferon (IFN-γ) responses, and were found to be more suitable for immunotherapy. Seven key DE-FRGs with prognostic value were found to be considerably influenced by different chemotherapy drugs. Conclusion: A prognostic model based on seven DE-FRGs may efficiently predict patient prognosis and immunotherapy response, helping to develop individualized treatment strategies in ESCA

c-Jun N-terminal kinase (JNK) is required for survival and proliferation of B-lymphoma cells

Several primary murine and human B lymphomas and cell lines were found to constitutively express high levels of the activated form of c-jun N-terminal kinase (JNK), a member of the mitogen-activated protein (MAP) kinase family. Proliferation of murine B lymphomas CH31, CH12.Lx, BKS-2, and WEHI-231 and the human B lymphomas BJAB, RAMOS, RAJI, OCI-Ly7, and OCI-Ly10 was strongly inhibited by SP600125, an anthrapyrazolone inhibitor of JNK, in a dose-dependent manner. The lymphoma cells underwent apoptosis and arrested at the G2/M phase of cell cycle. Furthermore, JNK-specific small interfering RNA (siRNA) inhibited the growth of both murine and human B lymphomas. Thus in the B-lymphoma model, JNK appears to have a unique prosurvival role. Survival signals provided by CD40 and interleukin-10 (IL-10) together reversed the growth inhibition induced by the JNK inhibitor. c-Myc protein levels were reduced in the presence of both SP600125 and JNK-specific siRNA, and CD40 ligation restored c-Myc levels. Moreover, Bcl-xL rescued WEHI-231 cells from apoptosis induced by the JNK inhibitor. The JNK inhibitor also reduced levels of early growth response gene-1 (Egr-1) protein, and overexpressing Egr-1 partially rescued lymphoma cells from apoptosis. Thus, JNK may act via c-Myc and Egr-1, which were shown to be important for B-lymphoma survival and growth. (Blood. 2005;106:1382-1391