The cGAS-STING signaling in cardiovascular and metabolic diseases: Future novel target option for pharmacotherapy.

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling exert essential regulatory function in microbial-and onco-immunology through the induction of cytokines, primarily type I interferons. Recently, the aberrant and deranged signaling of the cGAS-STING axis is closely implicated in multiple sterile inflammatory diseases, including heart failure, myocardial infarction, cardiac hypertrophy, nonalcoholic fatty liver diseases, aortic aneurysm and dissection, obesity, etc. This is because of the massive loads of damage-associated molecular patterns (mitochondrial DNA, DNA in extracellular vesicles) liberated from recurrent injury to metabolic cellular organelles and tissues, which are sensed by the pathway. Also, the cGAS-STING pathway crosstalk with essential intracellular homeostasis processes like apoptosis, autophagy, and regulate cellular metabolism. Targeting derailed STING signaling has become necessary for chronic inflammatory diseases. Meanwhile, excessive type I interferons signaling impact on cardiovascular and metabolic health remain entirely elusive. In this review, we summarize the intimate connection between the cGAS-STING pathway and cardiovascular and metabolic disorders. We also discuss some potential small molecule inhibitors for the pathway. This review provides insight to stimulate interest in and support future research into understanding this signaling axis in cardiovascular and metabolic tissues and diseases.

Shenmai Injection Protects Against Doxorubicin-Induced Cardiotoxicity via Maintaining Mitochondrial Homeostasis.

Shenmai injection (SMI), as a patented traditional Chinese medicine, is extracted from Panax ginseng and Ophiopogon japonicus. It commonly used in the treatment of cardiovascular disease and in the control of cardiac toxicity induced by doxorubicin (DOX) treatment. However, its anti-cardiotoxicity mechanism remains unknown. The purpose of this study was to investigate the underlying mitochondrial protective mechanisms of SMI on DOX-induced myocardial injury. The cardioprotective effect of SMI against DOX-induced myocardial damage was evaluated in C57BL/6 mice and H9c2 cardiomyocytes. In vivo, myocardial injury, apoptosis and phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB/Akt)/glycogen synthase kinase 3 beta (GSK-3ß) signaling pathway related proteins were measured. In vitro, apoptosis, mitochondrial superoxide, mitochondrial membrane potential, mitochondrial morphology, levels of mitochondrial fission/fusion associated proteins, mitochondrial respiratory function, and AMP-activated protein kinase (AMPK) activity were assessed. To further elucidate the regulating effects of SMI on AMPK and PI3K/Akt/GSK-3ß signaling pathway, compound C and LY294002 were utilized. In vivo, SMI decreased mortality rate, levels of creatine kinase, and creatine kinase-MB. SMI significantly prevented DOX-induced cardiac dysfunction and apoptosis, decreased levels of Bax/Bcl-2 and cleaved-Caspase3, increased levels of PI3K, p-Akt, and p-GSK-3ß. In vitro, SMI rescued DOX-injured H9c2 cardiomyocytes from apoptosis, excessive mitochondrial reactive oxygen species production and descending mitochondrial membrane potential, which were markedly suppressed by LY294002. SMI increased ratio of L-OPA1 to S-OPA1, levels of AMPK phosphorylation, and DRP1 phosphorylation (Ser637) in order to prevent DOX-induced excessive mitochondrial fission and insufficient mitochondrial fusion. In conclusion, SMI prevents DOX-induced cardiotoxicity, inhibits mitochondrial oxidative stress and mitochondrial fragmentation through activation of AMPK and PI3K/Akt/GSK-3ß signaling pathway.

Differential expression profile analysis of cisplatin­regulated miRNAs in a human gastric cancer cell line.

Cisplatin, one of the most commonly used drugs in combination chemotherapy, is an effective anti­tumor agent widely used for diverse tumor types. MicroRNAs (miRNAs/miRs) are involved in the occurrence, development, diagnosis and treatment of cancer. Therefore, the aim of the current study was to explore whether cisplatin exerts anticancer effects by causing differential expression of miRNAs in human gastric cancer cells. The human gastric cancer cell line NCI­N87 was cultured with a certain dose of cisplatin and high­throughput sequencing combined with reverse transcription­quantitative polymerase chain reaction (RT­qPCR) was performed to detect cisplatin­regulated miRNAs. miRNAs upregulated and downregulated following cisplatin exposure were analyzed. High­throughput sequencing revealed 33 upregulated and 16 downregulated miRNAs. A total of five significantly upregulated and five significantly downregulated miRNAs were identified by RT­qPCR. The expression levels of hsa­miR­1246 and hsa­miR­892b were consistent with the results obtained from high­throughput sequencing. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway clustering of cisplatin­regulated miRNAs revealed that the miRNAs regulated genes involved in several biological processes and signaling pathways. The results obtained in the current study suggested that cisplatin may exert an important anticancer effect in gastric cancer via complex biological processes and signaling pathways.

Combination of Enhanced Depth Imaging Optical Coherence Tomography and Fundus Images for Glaucoma Screening.

Glaucoma is an eye disease that damages the optic nerve and can lead to irreversible loss of peripheral vision gradually and even blindness without treatment. Thus, diagnosing glaucoma in the early stage is essential for treatment. In this paper, an automatic method for early glaucoma screening is proposed. The proposed method combines structural parameters and textural features extracted from enhanced depth imaging optical coherence tomography (EDI-OCT) images and fundus images. The method first segments anterior the lamina cribrosa surface (ALCS) based on region-aware strategy and residual U-Net and then extracts structural features of the lamina cribrosa, such as lamina cribrosa depth and deformation of lamina cribrosa. In fundus images, scanning lines based on disc center and brightness reduction are used for optic disc segmentation and brightness compensation is utilized for segmenting the optic cup. Afterward, the cup-to-disc ratio (CDR) and textural features are extracted from fundus images. Hybrid features are used for training and classification to screen glaucoma by gcForest in the early stage. The proposed method has given exceptional results with 96.88% accuracy and 91.67% sensitivity.

Characterization of the Selective Indoleamine 2,3-Dioxygenase-1 (IDO1) Catalytic Inhibitor EOS200271/PF-06840003 Supports IDO1 as a Critical Resistance Mechanism to PD-(L)1 Blockade Therapy.

Tumors use indoleamine 2,3-dioxygenase-1 (IDO1) as a major mechanism to induce an immunosuppressive microenvironment. IDO1 expression is upregulated in many cancers and considered to be a resistance mechanism to immune checkpoint therapies. IDO1 is induced in response to inflammatory stimuli such as IFNγ and promotes immune tolerance by depleting tryptophan and producing tryptophan catabolites, including kynurenine, in the tumor microenvironment. This leads to effector T-cell anergy and enhanced Treg function through upregulation of FoxP3. As a nexus for the induction of key immunosuppressive mechanisms, IDO1 represents an important immunotherapeutic target in oncology. Here, we report the identification and characterization of the novel selective, orally bioavailable IDO1 inhibitor EOS200271/PF-06840003. It reversed IDO1-induced T-cell anergy in vitro In mice carrying syngeneic tumor grafts, PF-06840003 reduced intratumoral kynurenine levels by over 80% and inhibited tumor growth both in monotherapy and, with an increased efficacy, in combination with antibodies blocking the immune checkpoint ligand PD-L1. We demonstrate that anti-PD-L1 therapy results in increased IDO1 metabolic activity thereby providing additional mechanistic rationale for combining PD-(L)1 blockade with IDO1 inhibition in cancer immunotherapies. Supported by these preclinical data and favorable predicted human pharmacokinetic properties of PF-06840003, a phase I open-label, multicenter clinical study (NCT02764151) has been initiated.

Phase I study of glasdegib (PF-04449913), an oral smoothened inhibitor, in Japanese patients with select hematologic malignancies.

The hedgehog signaling pathway regulates multiple morphogenetic processes during embryogenesis. Aberrant activation of the hedgehog pathway signal transduction in adult tissues is associated with the pathogenesis of hematologic malignancies and solid tumors. We report findings from an open-label, multicenter phase I trial of the selective, small-molecule hedgehog signaling inhibitor glasdegib (PF-04449913) in Japanese patients with select advanced hematologic malignancies. Glasdegib was administered as once-daily oral doses (25, 50 and 100 mg) in 28-day cycles after a lead-in dose on Day -5. The primary objectives were to determine first-cycle dose-limiting toxicities, safety, vital signs and laboratory test abnormalities. Secondary objectives included evaluation of pharmacokinetics, pharmacodynamics and preliminary evidence of clinical activity of glasdegib. No dose-limiting toxicities were noted in the 13 patients in the present study. All patients experienced at least one treatment-emergent, all-causality adverse event. The most frequent treatment-related adverse events (observed in ≥3 patients) were dysgeusia (n = 9), muscle spasms (n = 5), alopecia, decreased appetite (n = 4 each), and increased blood creatinine phosphokinase, constipation and diarrhea (n = 3 each). Two deaths occurred during the study and were deemed not to be treatment-related due to disease progression. Glasdegib demonstrated dose-proportional pharmacokinetics, marked downregulation of the glioma-associated transcriptional regulator GLI1 expression in normal skin, and evidence of preliminary clinical activity, although data are limited. Glasdegib was safe and well tolerated across the dose levels tested. It is confirmed that the 100-mg dose is safe and tolerable in Japanese patients, and this dose level will be examined in the future clinical trial.

Bilateral Effects of Excipients on Protein Stability: Preferential Interaction Type of Excipient and Surface Aromatic Hydrophobicity of Protein.

PURPOSE: Understanding the mechanism of protein-excipient interaction and illuminating the influencing factors on protein stability are key steps in the rational design of protein formulations. The objective of this study was to assess effects of preferential interaction type of excipient and surface aromatic hydrophobicity of protein on protein solution stability. METHODS: The preferential interaction between excipient and aromatic hydrophobic area of protein was investigated by solubility and fluorescence studies of amino acid derivatives in excipient solutions. We examined conformational, colloidal and mechanical stabilities of model proteins with different surface aromatic hydrophobicities, including bovine serum albumin (BSA) and ovalbumin (OVA), and then stability data were visualized by three-index empirical phase diagram. RESULTS: The result showed that preferentially excluded excipients (trehalose, sucrose and sorbitol) protected protein conformation against damage, but they could accelerate mechanical stress-induced aggregation. Preferentially bound excipients (propanediol and arginine) suppressed BSA aggregation, but arginine failed to inhibit OVA aggregation, which might be attributed to the disparate conformational perturbing effects of arginine on aromatic hydrophobic regions of BSA and OVA. CONCLUSIONS: These findings provided strong evidence that excipient possessed bilateral effects, and its application should be determined on different preferential interaction behaviors of excipients with protein, especially with the aromatic hydrophobic region.

A NOTCH1 gene copy number gain is a prognostic indicator of worse survival and a predictive biomarker to a Notch1 targeting antibody in colorectal cancer.

Dysregulation of the Notch1 receptor has been shown to facilitate the development and progression of colorectal cancer (CRC) and has been identified as an independent predictor of disease progression and worse survival. Although mutations in the NOTCH1 receptor have not been described in CRC, we have previously discovered a NOTCH1 gene copy number gain in a portion of CRC tumor samples. Here, we demonstrated that a NOTCH1 gene copy number gain is significantly associated with worse survival and a high percentage of gene duplication in a cohort of patients with advanced CRC. In our CRC patient-derived tumor xenograft (PDTX) model, tumors harboring a NOTCH1 gain exhibited significant elevation of the Notch1 receptor, JAG1 ligand and cleaved Notch1 activity. In addition, a significant association was identified between a gain in NOTCH1 gene copy number and sensitivity to a Notch1-targeting antibody. These findings suggest that patients with metastatic CRC that harbor a gain in NOTCH1 gene copy number have worse survival and that targeting this patient population with a Notch1 antibody may yield improved outcomes.

Treatment with PF-04449913, an oral smoothened antagonist, in patients with myeloid malignancies: a phase 1 safety and pharmacokinetics study.

BACKGROUND: Activation of the Hedgehog signalling pathway contributes to cancer progression and the development of myeloid leukaemia stem cell therapeutic resistance. We aimed to identify the maximum tolerated dose (MTD) and the recommended phase 2 dose of the selective Hedgehog antagonist PF-04449913 in myeloid malignancies. METHODS: We undertook an open-label, dose-finding, standard 3+3 design phase 1 study of PF-04449913 in adult patients with acute myeloid leukaemia, chronic myeloid leukaemia, chronic myelomonocytic leukaemia, myelodysplastic syndrome, or myelofibrosis who were refractory, resistant, or intolerant to previous treatments, at three centres in the USA and one in Italy. Patients who had newly diagnosed, untreated disease were included if they were not eligible for standard treatment options or if standard treatments were not deemed appropriate. Patients received PF-04449913 once daily continuously until disease progression, unacceptable toxic effects, or patient withdrawal for up to 12 28-day cycles. Additional cycles were given if patients showed evidence of clinical benefit. The starting dose was 5 mg and was increased by 100% until the first dose-limiting toxic effect (DLT) and by 50% thereafter, in keeping with a 3+3 clinical trial statistical design. The primary endpoint was first-cycle DLTs. Secondary endpoints were safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary clinical activity. This trial is registered with ClinicalTrials.gov, number NCT00953758. FINDINGS: Between March 24, 2010, and Sept 7, 2012, 47 patients were enrolled and included in the study: 28 with acute myeloid leukaemia, six with myelodysplastic syndrome, five with chronic myeloid leukaemia (two with chronic-phase and three with blast-phase disease), one with chronic myelomonocytic leukaemia, and seven with myelofibrosis. Patients received PF-04449913 once daily at 5 mg (n=3), 10 mg (n=3), 20 mg (n=4), 40 mg (n=4), 80 mg (n=8), 120 mg (n=3), 180 mg (n=3), 270 mg (n=5), 400 mg (n=9), and 600 mg (n=5). Two patients experienced DLTs (one each in the 80 mg and 600 mg dose groups). The MTD for PF-04449913 was established to be 400 mg once daily. Of the 47 patients enrolled, 28 (60%) experienced treatment-related adverse events, three of which were grade 4 in severity. The most common treatment-related adverse events included dysgeusia (13 [28%] patients), decreased appetite (nine [19%]), and alopecia (seven [15%]). None of the 15 deaths reported were treatment related. Pharmacokinetics seemed to be dose proportional. The mean half-life was 23·9 h (SD 14·0) in the MTD group. Some suggestion of clinical activity was noted in 23 (49%) of 47 patients with haematological malignancies. Based on these results, the recommended phase 2 dose was 200 mg or lower once daily. INTERPRETATION: Based on these findings, PF-04449913 is being tested in phase 2 studies in patients with myelodysplastic syndrome, acute myeloid leukaemia, and myelofibrosis. FUNDING: Pfizer.

PEST domain mutations in Notch receptors comprise an oncogenic driver segment in triple-negative breast cancer sensitive to a γ-secretase inhibitor.

PURPOSE: To identify and characterize novel, activating mutations in Notch receptors in breast cancer and to determine response to the gamma secretase inhibitor (GSI) PF-03084014. EXPERIMENTAL DESIGN: We used several computational approaches, including novel algorithms, to analyze next-generation sequencing data and related omic datasets from The Cancer Genome Atlas (TCGA) breast cancer cohort. Patient-derived xenograft (PDX) models were sequenced, and Notch-mutant models were treated with PF-03084014. Gene-expression and functional analyses were performed to study the mechanism of activation through mutation and inhibition by PF-03084014. RESULTS: We identified mutations within and upstream of the PEST domains of NOTCH1, NOTCH2, and NOTCH3 in the TCGA dataset. Mutations occurred via several genetic mechanisms and compromised the function of the PEST domain, a negative regulatory domain commonly mutated in other cancers. Focal amplifications of NOTCH2 and NOTCH3 were also observed, as were heterodimerization or extracellular domain mutations at lower incidence. Mutations and amplifications often activated the Notch pathway as evidenced by increased expression of canonical Notch target genes, and functional mutations were significantly enriched in the triple-negative breast cancer subtype (TNBC). PDX models were also identified that harbored PEST domain mutations, and these models were highly sensitive to PF-03084014. CONCLUSIONS: This work suggests that Notch-altered breast cancer constitutes a bona fide oncogenic driver segment with the most common alteration being PEST domain mutations present in multiple Notch receptors. Importantly, functional studies suggest that this newly identified class can be targeted with Notch inhibitors, including GSIs.

A Phase I, dose-finding study in patients with advanced solid malignancies of the oral γ-secretase inhibitor PF-03084014.

PURPOSE: To estimate the maximum tolerated dose (MTD) for continuous oral administration of the γ-secretase inhibitor PF-03084014, determine the recommended phase II dose (RP2D), and evaluate safety and preliminary activity in patients with advanced solid tumors. EXPERIMENTAL DESIGN: This open-label, phase I study consisted of a dose-finding portion based on a 3+3 design, followed by an expansion cohort. PF-03084014 was administered orally, twice daily (BID) for 21 continuous days. Tested doses ranged from 20 to 330 mg BID. In the expansion cohort, patients were to receive the estimated MTD or a lower dose of PF-03084014. RESULTS: A total of 64 patients received treatment. The MTD was estimated to be 220 mg BID. The RP2D was determined to be 150 mg BID, based on the better safety profile versus the 220-mg BID dose, given comparable NOTCH-related target inhibition. The most common treatment-related adverse events were diarrhea, nausea, fatigue, hypophosphatemia, vomiting, rash, and decreased appetite, which were generally mild to moderate in severity. One patient with advanced thyroid cancer had a complete response, and five of seven response-evaluable patients with desmoid tumor achieved a partial response (71.4% objective response rate). Tumor responses were mostly durable, ranging from 1.74+ to 24+ months. PF-03084014 demonstrated a generally dose-dependent pharmacokinetic profile at doses ranging from 20 to 330 mg BID. Consistent downmodulation of NOTCH-related HES4 gene expression was observed in peripheral blood from all evaluable patients. CONCLUSION: Further development of PF-03084014 for the treatment of patients with advanced solid tumors is warranted and currently under evaluation.

A phase I study of PF-04449913, an oral hedgehog inhibitor, in patients with advanced solid tumors.

PURPOSE: To estimate the maximum tolerated dose (MTD) of single-agent PF-04449913, and to evaluate safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity in patients with advanced tumors. EXPERIMENTAL DESIGN: A 3+3 design was used in this open-label, multicenter, phase I study and dose escalation/de-escalation applied until identification of the MTD. PF-04449913 was orally administered once daily in continuous 28-day treatment cycles. The starting dose was 80 mg. RESULTS: A total of 23 patients were enrolled; 19 were evaluable for first-cycle dose-limiting toxicity (DLT). The first-cycle DLT rate at the 640 mg dose level was 33.3%, and the MTD was estimated to be 320 mg once daily. The recommended phase II dose was not determined. PF-04449913 was generally well tolerated at doses of 80 to 320 mg once daily. The most common treatment-related adverse events (AE) were grade 1-2 dysgeusia, fatigue, decreased appetite, nausea, dizziness, dehydration, and diarrhea. Treatment-related grade 3 AEs only occurred in patients receiving PF-04449913 640 mg once daily. No treatment-related grade 4-5 AEs were reported. Pharmacokinetic analysis indicated a generally dose-proportional kinetics with biphasic elimination, supporting once-daily dosing. PF-04449913 modulated hedgehog signaling at the dose levels tested, as demonstrated by >80% downregulation of GLI1 expression in the skin of treated patients. Eight patients (34.8%) achieved stable disease; none had complete or partial response. Three patients with disease progression at enrollment had prolonged disease stabilization (≥6 months). CONCLUSIONS: The results obtained in this study support further evaluation of PF-04449913 in patients with advanced solid tumors.

Preferential interactions between protein and arginine: effects of arginine on tertiary conformational and colloidal stability of protein solution.

The purpose of this study was to better understand the preferential binding behavior of arginine to protein as well as the impact of arginine on the conformational and colloidal stability of protein solution. Physical stabilities of model proteins, bovine serum albumin (BSA) and ovalbumin (OVA), were investigated by fluorescence-based and dynamic light scattering techniques in the absence and presence of arginine. We investigated the interactions between arginine and tryptophan or tyrosine residues by conducting solubility and fluorescence studies of two amino acid derivatives, N-acetyl-l-tryptophanamide (NATA) and N-acetyl-l-tyrosinamide (NAYA), in arginine solutions. The result showed that arginine preferentially bond to the aromatic amino acids of proteins mainly through hydrogen bonds and Van der Waals' forces, while the binding constant K of arginine with BSA and OVA at 298K was 41.92 and 5.77L/mol, respectively. The fluorescence quenching, the decreased fluorescence lifetime and the red-shifted ANS peak position revealed that arginine perturbed the local environment of tryptophan and tyrosine residues. We also found the attenuated electrostatic repulsion among BSA and OVA molecules after adding arginine. These findings provided strong evidence that arginine possessed negative effects on tertiary conformational and colloidal stability of BSA and OVA during the preferential binding process.

Molecular predictors of sensitivity to the insulin-like growth factor 1 receptor inhibitor Figitumumab (CP-751,871).

Figitumumab (CP-751,871), a potent and fully human monoclonal anti-insulin-like growth factor 1 receptor (IGF1R) antibody, has been investigated in clinical trials of several solid tumors. To identify biomarkers of sensitivity and resistance to figitumumab, its in vitro antiproliferative activity was analyzed in a panel of 93 cancer cell lines by combining in vitro screens with extensive molecular profiling of genomic aberrations. Overall response was bimodal and the majority of cell lines were resistant to figitumumab. Nine of 15 sensitive cell lines were derived from colon cancers. Correlations between genomic characteristics of cancer cell lines with figitumumab antiproliferative activity revealed that components of the IGF pathway, including IRS2 (insulin receptor substrate 2) and IGFBP5 (IGF-binding protein 5), played a pivotal role in determining the sensitivity of tumors to single-agent figitumumab. Tissue-specific differences among the top predictive genes highlight the need for tumor-specific patient selection strategies. For the first time, we report that alteration or expression of the MYB oncogene is associated with sensitivity to IGF1R inhibitors. MYB is dysregulated in hematologic and epithelial tumors, and IGF1R inhibition may represent a novel therapeutic opportunity. Although growth inhibitory activity with single-agent figitumumab was relatively rare, nine combinations comprising figitumumab plus chemotherapeutic agents or other targeted agents exhibited properties of synergy. Inhibitors of the ERBB family were frequently synergistic and potential biomarkers of drug synergy were identified. Several biomarkers of antiproliferative activity of figitumumab both alone and in combination with other therapies may inform the design of clinical trials evaluating IGF1R inhibitors.

Biomarker and pharmacologic evaluation of the γ-secretase inhibitor PF-03084014 in breast cancer models.

PURPOSE: We aimed to assess the biologic activity of PF-03084014 in breast xenograft models. The biomarkers for mechanism and patient stratification were also explored. EXPERIMENTAL DESIGN: The in vitro and in vivo properties of PF-03084014 were investigated. The mRNA expressions of 40 key Notch pathway genes at baseline or after treatment were analyzed to link with the antitumor efficacy of PF-03084014 in a panel of breast cancer xenograft models. RESULTS: In vitro, PF-03084014 exhibited activity against tumor cell migration, endothelial cell tube formation, and mammosphere formation. In vivo, we observed apoptosis, antiproliferation, reduced tumor cell self-renewal ability, impaired tumor vasculature, and decreased metastasis activity after the treatment of PF-03084014. PF-03084014 treatment displayed significant antitumor activity in 10 of the 18 breast xenograft models. However, the antitumor efficacy in most models did not correlate with the in vitro antiproliferation results in the corresponding cell lines, suggesting the critical involvement of tumor microenvironment during Notch activation. In the tested breast xenograft models, the baseline expressions of the Notch receptors, ligands, and the cleaved Notch1 failed to predict the antitumor response to PF-03084014, whereas several Notch pathway target genes, including HEY2, HES4, and HES3, strongly corresponded with the response with a P value less than 0.01. Many of the best molecular predictors of response were also significantly modulated following PF-03084014 treatment. CONCLUSIONS: PF-03084014 showed antitumor and antimetastatic properties via pleiotropic mechanisms. The Notch pathway downstream genes may be used to predict the antitumor activity of PF-03084014 and enrich for responders among breast cancer patients.

An integrated genomic approach to identify predictive biomarkers of response to the aurora kinase inhibitor PF-03814735.

PF-03814735 is a novel, reversible inhibitor of Aurora kinases A and B that finished a phase I clinical trial for the treatment of advanced solid tumors. To find predictive biomarkers of drug sensitivity, we screened a diverse panel of 87 cancer cell lines for growth inhibition upon PF-03814735 treatment. Small cell lung cancer (SCLC) and, to a lesser extent, colon cancer lines were very sensitive to PF-03814735. The status of the Myc gene family and retinoblastoma pathway members significantly correlated with the efficacy of PF-03814735. Whereas RB1 inactivation, intact CDKN2A/p16, and normal CCND1/Cyclin D1 status are hallmarks of SCLC, activation or amplification of any of the three Myc genes (MYC, MYCL1, and MYCN) clearly differentiated cell line sensitivity within the SCLC panel. By contrast, we found that expression of Aurora A and B were weak predictors of response. We observed a decrease in histone H3 phosphorylation and polyploidization of sensitive lines, consistent with the phenotype of Aurora B inhibition. In vivo experiments with two SCLC xenograft models confirmed the sensitivity of Myc gene-driven models to PF-03814735 and a possible schedule dependence of MYC/c-Myc-driven tumors. Altogether our results suggest that SCLC and other malignancies driven by the Myc family genes may be suitable indications for treatment by Aurora B kinase inhibitors.

[Research on the methods for titrating respiratory syncytial virus].

OBJECTIVE: Respiratory syncytial virus (RSV) is the most common cause of lower respiratory infection in infants. It is very important to quantitative assay of RSV titer in the study on RSV pathogenesis, candidate vaccine and antiviral treatment. Therefore, we develop Real-time Quantitative PCR (Q-PCR) assay and enzyme immunospots (EIS) for titrating RSV and compare them with traditional 50% tissue culture infectious doses (TCID50). METHODS: Q-PCR, based upon the RSV-L genes, and EIS were utilized to titrate samples from RSV culture supernatants and RSV infected mouse lungs. Then, the results were compared with TCID50. RESULTS: For the samples from RSV culture supernatants, the ratio of Q-PCR and EIS (plaque forming unit, pfu) was 10:1 and the ratio of EIS and TCID50 was 10:1 when TCID50 was converted as pfu. For the samples from RSV infected mouse lungs, the ratio of Q-PCR and EIS was 1000:1 and the ratio of EIS and TCID50 was 5:1. CONCLUSION: We have successfully established Q-PCR and EIS to titrate RSV and compared them with TCID50. We concluded EIS is a cost-effective method to titrate RSV.

[In vitro transgenic expression efficacy of a helper-dependent adenoviral vector encoding enhanced green fluorescent protein].

To investigate the transgenic expressing efficacy of helper-dependent adenoviral vector (HDAd) in vitro, we constructed a HDAd encoding enhanced green fluorescent protein (EGFP), denominated as HDAd/EGFP, performed large scale preparation and purification, and then identified the purified HDAd/EGFP under fluorescent microscope and electron microscope. After the concentration of HDAd/EGFP was determined by spectrophotometer, the transgenic expression efficiency of HDAd/EGFP was compared with first generation adenoviral vector encoding EGFP (FGAd/EGFP) in vitro. Therefore, we infected A549 cells with 2000 virus particles (vp) per cell by HDAd/EGFP and FGAd/EGFP respectively and analyzed EGFP expressing level by flow cytometry. Consequently, the fluorescent expression rate and fluorescent intensity of EGFP were higher in early infected A549 cells by HDAd/EGFP than by FGAd/EGFP. HDAd, capable of expressing transgene instantly and efficiently in vitro, is a potential vaccine vector.

A prime-boost vaccination strategy using attenuated Salmonella typhimurium and a replication-deficient recombinant adenovirus vector elicits protective immunity against human respiratory syncytial virus.

Human respiratory syncytial virus (RSV), for which no clinically approved vaccine is available yet, is globally a serious pediatric pathogen of the lower respiratory tract. Several approaches have been used to develop vaccines against RSV, but none of these have been approved for use in humans. An efficient vaccine-enhancing strategy for RSV is still urgently needed. We found previously that oral SL7207/pcDNA3.1/F and intranasal FGAd/F were able to induce an effective protective immune response against RSV. The heterologous prime-boost immunization regime has been reported recently to be an efficient vaccine-enhancing strategy. Therefore, we investigated the ability of an oral SL7207/pcDNA3.1/F prime and intranasal (i.n.) FGAd/F boost regimen to generate immune responses to RSV. The SL7207/pcDNA3.1/F prime-FGAd/F boost regimen generated stronger RSV-specific humoral and mucosal immune responses in BALB/c mice than the oral SL7207/pcDNA3.1/F regimen alone, and stronger specific cellular immune responses than the i.n. FGAd/F regimen alone. Histopathological analysis showed an increased efficacy against RSV challenge by the heterologous prime-boost regimen. These results suggest that such a heterologous prime-boost strategy can enhance the efficacy of either the SL7207 or the FGAd vector regimen in generating immune responses in BALB/c mice.

Intranasal vaccination with a helper-dependent adenoviral vector enhances transgene-specific immune responses in BALB/c mice.

Helper-dependent adenoviral (HDAd) vectors were developed primarily for genetic disease therapy by deleting all coding regions for attenuating the host cellular immune response to adenovirus (Ad) and long-lasting gene expression. Recently Harui et al. reported that HDAd vaccine could stimulate superior transgene-specific cytotoxic T lymphocyte (CTL) and antibody responses via the intraperitoneal route, compared to first-generation adenoviral (FGAd) vaccine. This prompted us to explore the potential of HDAd as a vaccine vector administrated intranasally. In this study, we prepared HDAd and FGAd vectors expressing enhanced green fluorescent protein (EGFP), respectively, and compared their efficacy in mice. Mice were immunized intranasally with 5x10(9) vp HDAd or FGAd vector particles. Despite stimulating similar anti-Ad antibody responses with FGAd vaccine in the prime/boost strategy, HDAd vector expressing EGFP displayed superior transgene-specific serum IgG, mucosal IgA and cellular immune response, with the characterization of balanced or mixed Th1/Th2 CD4+ T-cell responses. Meanwhile, a single dose of intranasal (i.n.) vaccine of HDAd-EGFP induced a serum IgG response with more efficacy than FGAd-EGFP. In addition, i.n. boost immunization enhanced transgene-specific humoral and cellular responses, compared to single i.n. HDAd-EGFP immunization. Our results suggest that HDAd has potential for a mucosal vaccine vector via i.n. route, which will be useful for the development of vaccines against respiratory viruses, such as respiratory syncytial virus and influenza virus.