Apoptosis is an innate cellular defense response to viral infection. The slow-replicating human cytomegalovirus (HCMV) blocks premature death of host cells prior to completion of the infection cycle. In this study, we report that the HCMV UL38 gene encodes a cell death inhibitory protein. A mutant virus lacking the pUL38 coding sequence, ADdlUL38, grew poorly in human fibroblasts, failed to accumulate viral DNA to wild-type levels, and induced excessive death of infected cells. Cells expressing pUL38 were resistant to cell death upon infection and effectively supported the growth of ADdlUL38. Cells infected with the pUL38-deficient virus showed morphological changes characteristic of apoptosis, including cell shrinkage, membrane blebbing, vesicle release, and chromatin condensation and fragmentation. The proteolytic cleavage of two key enzymes involved in apoptosis, namely, caspase 3 and poly(ADP-ribose) polymerase, was activated upon ADdlUL38 infection, and the cleavage was blocked in cells expressing pUL38. The pan-caspase inhibitor Z-VAD-FMK largely restored the growth of ADdlUL38 in normal fibroblasts, indicating that the defective growth of the mutant virus mainly resulted from premature death of host cells. Furthermore, cells expressing pUL38 were resistant to cell death induced by a mutant adenovirus lacking the antiapoptotic E1B-19K protein or by thapsigargin, which disrupts calcium homeostasis in the endoplasmic reticulum. Taken together, these results indicate that the HCMV protein pUL38 suppresses apoptosis, blocking premature death of host cells to facilitate efficient virus replication.

LRRC8 volume-regulated anion channels transmit cGAMP into the bystander cells for interferon response and reinforcement of anti-viral defense

The endoplasmic reticulum (ER) is a key organelle involved in sensing and responding to stressful conditions, including those resulting from infection of viruses, such as human cytomegalovirus (HCMV). Three signaling pathways collectively termed the unfolded protein response (UPR) are activated to resolve ER stress, but they will also lead to cell death if the stress cannot be alleviated. HCMV is able to modulate the UPR to promote its infection. The specific viral factors involved in such HCMV-mediated modulation, however, were unknown. We previously showed that HCMV protein pUL38 was required to maintain the viability of infected cells, and it blocked cell death induced by thapsigargin. Here, we report that pUL38 is an HCMV-encoded regulator to modulate the UPR. In infection, pUL38 allowed HCMV to upregulate phosphorylation of PKR-like ER kinase (PERK) and the ␣ subunit of eukaryotic initiation factor 2 (eIF-2␣), as well as induce robust accumulation of activating transcriptional factor 4 (ATF4), key components of the PERK pathway. pUL38 also allowed the virus to suppress persistent phosphorylation of c-Jun N-terminal kinase (JNK), which was induced by the inositol-requiring enzyme 1 pathway. In isolation, pUL38 overexpression elevated eIF-2␣ phosphorylation, induced ATF4 accumulation, limited JNK phosphorylation, and suppressed cell death induced by both thapsigargin and tunicamycin, two drugs that induce ER stress by different mechanisms. Importantly, ATF4 overexpression and JNK inhibition significantly reduced cell death in pUL38-deficient virus infection. Thus, pUL38 targets ATF4 expression and JNK activation, and this activity appears to be critical for protecting cells from ER stress induced by HCMV infection.

Methylation profiling of an 11-gene set in urine sediments provides a sensitive and specific detection of bladder cancer.

Purpose: To investigate the incidence of FGFR1 amplification in Chinese non-small cell lung cancer (NSCLC) and to preclinically test the hypothesis that the novel, potent, and selective fibroblast growth factor receptor (FGFR) small-molecule inhibitor AZD4547 will deliver potent antitumor activity in NSCLC FGFR1-amplified patient-derived tumor xenograft (PDTX) models.Experimental Design: A range of assays was used to assess the translational relevance of FGFR1 amplification and AZD4547 treatment including in vitro lung cell line panel screening and pharmacodynamic (PD) analysis, FGFR1 FISH tissue microarray (TMA) analysis of Chinese NSCLC (n ¼ 127), and, importantly, antitumor efficacy testing and PD analysis of lung PDTX models using AZD4547.Results: The incidence of FGFR1 amplification within Chinese patient NSCLC tumors was 12.5% of squamous origin (6 of 48) and 7% of adenocarcinoma (5 of 76). AZD4547 displayed a highly selective profile across a lung cell line panel, potently inhibiting cell growth only in those lines harboring amplified FGFR1 (GI 50 ¼ 0.003-0.111 mmol/L). AZD4547 induced potent tumor stasis or regressive effects in four of five FGFR1-amplified squamous NSCLC PDTX models. Pharmacodynamic modulation was observed in vivo, and antitumor efficacy correlated well with FGFR1 FISH score and protein expression level.Conclusions: This study provides novel epidemiologic data through identification of FGFR1 gene amplification in Chinese NSCLC specimens (particularly squamous) and, importantly, extends the clinical significance of this finding by using multiple FGFR1-amplified squamous lung cancer PDTX models to show tumor stasis or regression effects using a specific FGFR inhibitor (AZD4547). Thus, the translational science presented here provides a strong rationale for investigation of AZD4547 as a therapeutic option for patients with squamous NSCLC tumors harboring amplification of FGFR1. Clin Cancer Res; 18(24); 6658-67. Ó2012 AACR.

Rituximab (RTX), a chimeric anti-CD20 antibody, is associated with direct induction of apoptosis and antibody-dependent cell-mediated cytotoxicity (ADCC) with clinical efficacy in mantle cell lymphoma (MCL). Lenalidomide (LEN), a novel immunomodulatory agent, sensitizes tumor cells and enhances ADCC. Our study attempted to elucidate the mechanism of LEN-enhanced RTX-mediated cytotoxicity of MCL cells. We found that LEN and RTX induced growth inhibition of both cultured and fresh primary MCL cells. LEN enhanced RTX-induced apoptosis via upregulating phosphorylation of c-Jun N-terminal protein kinases (JNK), Bcl-2, Bad; increasing release of cytochrome-c; enhancing activation of caspase-3, -8, -9 and cleavage of PARP. Meanwhile, LEN activated NK cells and increased CD16 expression on CD56 low CD161 NK cells. Whole PBMCs but not NK cell-depleted PBMCs treated with LEN augmented 30% of RTX-dependent cytotoxicity. Daily treatment with LEN increased NK cells by 10-folds in SCID mice, and combination of LEN and RTX decreased tumor burden and prolonged survival of MCL-bearing SCID mice. Taken together, our study demonstrates that LEN plus RTX provides a synergistically therapeutic effect on MCL cells by enhancing apoptosis and RTX-dependent NK cell-mediated cytotoxicity and may be an optimal combination in the clinical trial of relapsed or refractory MCL. Am. J. Hematol. 84:553-559, 2009. V

In this study, we adopted a conditional protein genetic approach to characterize the role of the human cytomegalovirus (HCMV) gene UL79 during virus infection. We constructed ADddUL79, a recombinant HCMV in which the annotated UL79 open reading frame (ORF) was tagged with the destabilization domain of a highly unstable variant of the human FKBP12 protein (ddFKBP). The ddFKBP domain targets the tagged protein for rapid proteasomal degradation, but the synthetic ligand Shield-1 can stabilize ddFKBP, allowing accumulation of the tagged protein. ADddUL79 failed to replicate without Shield-1, but it grew at wild-type levels with Shield-1 or in human foreskin fibroblasts overexpressing hemagglutinin (HA)-tagged UL79 (HF-UL79HA cells), indicating an essential role of UL79 and the effectiveness of this approach. Without Shield-1, representative immediate-early and early viral proteins as well as viral DNA accumulated normally, but late transcripts and proteins were markedly reduced. UL79 was transcribed with early-late kinetics, which was also regulated via a positive-feedback loop. Using HF-UL79HA cells, we found that the UL79 protein localized to viral replication compartments during HCMV infection. Finally, we created a second UL79 mutant virus (ADinUL79 stop ) in which the UL79 ORF was disrupted by a stop codon mutation and found that ADinUL79 stop phenocopied ADddUL79 under the destabilizing condition. Taking these results together, we conclude that UL79 acts after viral DNA replication to promote the accumulation of late viral transcripts. Importantly, the comparative analysis of ADddUL79 and ADinUL79 stop viruses provide additional proof for the power of the protein stability-based conditional approach to dissect the role of viral factors in HCMV biology.

Background:Gastric cancer (GC) is a leading cause of cancer deaths worldwide. Since the approval of trastuzumab, targeted therapies are emerging as promising treatment options for the disease. This study aimed to explore the molecular segmentation of several known therapeutics targets, human epidermal growth factor receptor 2 (HER2), MET and fibroblast growth factor receptor 2 (FGFR2), within GC using clinically approved or investigational kits and scoring criteria. Knowledge of how these markers are segmented in the same cohort of GC patients could improve future clinical trial designs.Methods:Using immunohistochemistry (IHC) and FISH methods, overexpression and amplification of HER2, FGFR2 and MET were profiled in a cohort of Chinese GC samples. The correlations between anti-tumour sensitivity and the molecular segments of HER2, MET and FGFR2 alterations were further tested in a panel of GC cell lines and the patient-derived GC xenograft (PDGCX) model using the targeted inhibitors.Results:Of 172 GC patients, positivity for HER2, MET and FGFR2 alternations was found in 23 (13.4%), 21 (12.2%) and 9 (5.2%) patients, respectively. Positivity for MET was found in 3 of 23 HER2-positive GC patients. Co-positivity for FGFR2 and MET was found in 1 GC patient, and amplification of the two genes was found in different tumour cells. Our study in a panel of GC cell lines showed that in most cell lines, amplification or high expression of a particular molecular marker was mutually exclusive and in vitro sensitivity to the targeted agents lapatinib, PD173074 and crizotinib was only observed in cell lines with the corresponding high expression of the drugs' target protein. SGC031, an MET-positive PDGCX mouse model, responded to crizotinib but not to lapatinib or PD173074.Conclusions:Human epidermal growth factor receptor 2, MET and FGFR2 oncogenic driver alterations (gene amplification and overexpression) occur in three largely distinct molecular segments in GC. A significant proportion of HER2-negative patients may potentially benefit from MET- or FGFR2-targeted therapies.