In the fission yeast Schizosaccharomyces pombe, the protein kinase Chk1 has an essential role in transducing a delay signal to the cell cycle machinery in the presence of DNA damage. Fission yeast cells lacking the chk1 gene do not delay progression of the cell cycle in response to damage and are thus sensitive to DNA damaging agents. We have previously shown that Chk1 is phosphorylated following DNA damage induced by a variety of agents and that this is dependent on the integrity of the DNA damage checkpoint pathway, including Rad3, the ATR homolog. Through a combination of mutagenesis and phospho-specific antibodies, we have shown that serine at position 345 (S345) is phosphorylated in vivo in response to DNA damage, and that S345 phosphorylation is required for an intact checkpoint response. We have developed a kinase assay for Chk1, and have shown that basal Chk1 kinase activity is increased in response to DNA damage and that this increase, but not the basal activity, is dependent on S345. Furthermore, we show that S345 phosphorylation is required for Chk1 to associate with Rad24, a 14-3-3 protein, upon DNA damage. These results are consistent with a model whereby Chk1 phosphorylation results in increased Chk1 kinase activity that is necessary for both checkpoint delay and cellular survival following damage to the genome. These data are similar to observations made in mammalian cells and Xenopus oocyte extracts, suggesting that mechanisms leading to Chk1 activation have been conserved in evolution.

BackgroundSubstantial evidence suggests that the presence of inflammatory cells plays a critical role in the development and/or progression of human tumors. Neutrophils are the common inflammatory cells in tumors; however, the infiltration of intratumoral neutrophils in colorectal carcinoma (CRC) and its effect on CRC patients' prognosis are poorly understood.Methodology/Principal FindingsIn this study, the methods of tissue microarray and immunohistochemistry (IHC) were used to investigate the prognostic significance of intratumoral CD66b+ neutrophil in CRC. According to receiver operating characteristic curve analysis, the cutoff score for high intratumoral CD66b+ neutrophil in CRC was defined when the mean counts were more than 60 per TMA spot. In our study, high intratumoral CD66b+ neutrophil was observed in 104/229 (45.4%) of CRCs and in 29/229 (12.7%) of adjacent mucosal tissues. Further correlation analysis showed that high intratumoral neutrophil was positively correlated with pT status, pM status and clinical stage (P<0.05). In univariate survival analysis, a significant association between high intratumoral neutrophil and shortened patients' survival was found (P<0.0001). In different subsets of CRC patients, intratumoral neutrophil was also a prognostic indicator in patients with stage II, stage III, grade 2, grade 3, pT1, pT2, pN0 and pN1 (P<0.05). Importantly, high intratumoral neutrophil was evaluated as an independent prognostic factor in multivariate analysis (P<0.05).Conclusions/SignificanceOur results provide evidence that increased intratumoral neutrophil in CRC may be important in the acquisition of a malignant phenotype, indicating that the presence of intratumoral neutrophil is an independent factor for poor prognosis of patients with CRC.

Mature T-cell lymphomas, including peripheral T-cell lymphoma (PTCL) and extranodal NK/T-cell lymphoma (NKTL), represent a heterogeneous group of non-Hodgkin lymphomas with dismal outcomes and limited treatment options. To determine the extent of involvement of the JAK/STAT pathway in this malignancy, we performed targeted capture sequencing of 188 genes in this pathway in 171 PTCL and NKTL cases. A total of 272 nonsynonymous somatic mutations in 101 genes were identified in 73% of the samples, including 258 single-nucleotide variants and 14 insertions or deletions. Recurrent mutations were most frequently located in and (15%), followed by and (6%) and (4%). A high prevalence of mutation (21%) was observed specifically in NKTL. Novel mutations (p.D427H, E616G, p.E616K, and p.E696K) were shown to increase STAT3 phosphorylation and transcriptional activity of in the absence of cytokine, in which p.E616K induced programmed cell death-ligand 1 (PD-L1) expression by robust binding of activated STAT3 to the PD-L1 gene promoter. Consistent with these findings, PD-L1 was overexpressed in NKTL cell lines harboring hotspot mutations, and similar findings were observed by the overexpression of p.E616K and p.E616G in the wild-type NKTL cell line. Conversely, STAT3 silencing and inhibition decreased PD-L1 expression in mutant NKTL cell lines. In NKTL tumors, STAT3 activation correlated significantly with PD-L1 expression. We demonstrated that STAT3 activation confers high PD-L1 expression, which may promote tumor immune evasion. The combination of PD-1/PD-L1 antibodies and STAT3 inhibitors might be a promising therapeutic approach for NKTL, and possibly PTCL.

Our findings suggest that EZH2 protein, as examined by immunohistochemistry, may serve as a promising diagnostic biomarker of HCCs, and the use of a three-marker panel (EZH2, HSP70 and GPC3) can improve the rate of detection of HCCs in liver biopsy tissues.

Epitheliotropic intestinal T-cell lymphoma (EITL, also known as type II enteropathy-associated T-cell lymphoma) is an aggressive intestinal disease with poor prognosis and its molecular alterations have not been comprehensively characterized. We aimed to identify actionable easy-to-screen alterations that would allow better diagnostics and/or treatment of this deadly disease. By performing whole-exome sequencing of four EITL tumor-normal pairs, followed by amplicon deep sequencing of 42 tumor samples, frequent alterations of the JAK-STAT and G-protein-coupled receptor (GPCR) signaling pathways were discovered in a large portion of samples. Specifically, STAT5B was mutated in a remarkable 63% of cases, JAK3 in 35% and GNAI2 in 24%, with the majority occurring at known activating hotspots in key functional domains. Moreover, STAT5B locus carried copy-neutral loss of heterozygosity resulting in the duplication of the mutant copy, suggesting the importance of mutant STAT5B dosage for the development of EITL. Dysregulation of the JAK-STAT and GPCR pathways was also supported by gene expression profiling and further verified in patient tumor samples. In vitro overexpression of GNAI2 mutants led to the upregulation of pERK1/2, a member of MEK-ERK pathway. Notably, inhibitors of both JAK-STAT and MEK-ERK pathways effectively reduced viability of patient-derived primary EITL cells, indicating potential therapeutic strategies for this neoplasm with no effective treatment currently available.