Combination of PD-1 Inhibitor and OX40 Agonist Induces Tumor Rejection and Immune Memory in Mouse Models of Pancreatic Cancer.

BACKGROUND & AIMS: Advanced pancreatic ductal adenocarcinoma (PDAC) is resistant to therapy, including immune checkpoint inhibitors. We evaluated the effects of a neutralizing antibody against programmed cell death 1 (PD-1) and an agonist of OX40 (provides a survival signal to activated T cells) in mice with pancreatic tumors. METHODS: We performed studies in C57BL/6 mice (controls), KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) mice, and mice with orthotopic tumors grown from Panc02 cells, KrasG12D;P53flox/flox;PDX-1-Cre;Luciferase (KPC-Luc) cells, or mT4 cells. After tumors developed, mice were given injections of control antibody or anti-OX40 and/or anti-PD-1 antibody. Some mice were then given injections of antibodies against CD8, CD4, or NK1.1 to deplete immune cells, and IL4 or IL7RA to block cytokine signaling. Bioluminescence imaging was used to monitor tumor growth. Tumor tissues collected and single-cell suspensions were analyzed by time of flight mass spectrometry analysis. Mice that were tumor-free 100 days after implantation of orthotopic tumors were rechallenged with PDAC cells (KPC-Luc or mT4) and survival was measured. Median levels of PD-1 and OX40 mRNAs in PDACs were determined from The Cancer Genome Atlas and compared with patient survival times. RESULTS: In mice with orthotopic tumors, all those given control antibody or anti-PD-1 died within 50 days, whereas 43% of mice given anti-OX40 survived for 225 days; almost 100% of mice given the combination of anti-PD-1 and anti-OX40 survived for 225 days, and tumors were no longer detected. KPC mice given control antibody, anti-PD-1, or anti-OX40 had median survival times of 50 days or less, whereas mice given the combination of anti-PD-1 and anti-OX40 survived for a median 88 days. Mice with orthotopic tumors that were given the combination of anti-PD-1 and anti-OX40 and survived 100 days were rechallenged with a second tumor; those rechallenged with mT4 cells survived an additional median 70 days and those rechallenged with KPC-Luc cells survived long term, tumor free. The combination of anti-PD-1 and anti-OX40 did not slow tumor growth in mice with antibody-mediated depletion of CD4+ T cells. Mice with orthotopic tumors given the combination of anti-PD-1 and anti-OX40 that survived after complete tumor rejection were rechallenged with KPC-Luc cells; those with depletion of CD4+ T cells before the rechallenge had uncontrolled tumor growth. Furthermore, KPC orthotopic tumors from mice given the combination contained an increased number of CD4+ T cells that expressed CD127 compared with mice given control antibody. The combination of agents reduced the proportion of T-regulatory and exhausted T cells and decreased T-cell expression of GATA3; tumor size was negatively associated with numbers of infiltrating CD4+ T cells, CD4+CD127+ T cells, and CD8+CD127+ T cells, and positively associated with numbers of CD4+PD-1+ T cells, CD4+CD25+ T cells, and CD8+PD-1+ T cells. PDACs with high levels of OX40 and low levels of PD-1 were associated with longer survival times of patients. CONCLUSIONS: Pancreatic tumors appear to evade the immune response by inducing development of immune-suppressive T cells. In mice, the combination of anti-PD-1 inhibitory and anti-OX40 agonist antibodies reduces the proportion of T-regulatory and exhausted T cells in pancreatic tumors and increases numbers of memory CD4+ and CD8+ T cells, eradicating all detectable tumor. This information can be used in development of immune-based combination therapies for PDAC.

Protective effects of reduced dynamin-related protein 1 against amyloid beta-induced mitochondrial dysfunction and synaptic damage in Alzheimer's disease.

The purpose of our study was to understand the protective effects of reduced expression of dynamin-related protein (Drp1) against amyloid beta (Aß) induced mitochondrial and synaptic toxicities in Alzheimer's disease (AD) progression and pathogenesis. Our recent molecular and biochemical studies revealed that impaired mitochondrial dynamics-increased mitochondrial fragmentation and decreased fusion-in neurons from autopsy brains of AD patients and from transgenic AD mice and neurons expressing Aß, suggesting that Aß causes mitochondrial fragmentation in AD. Further, our recent co-immunoprecipitation and immunostaining analysis revealed that the mitochondrial fission protein Drp1 interacted with Aß, and this interaction increased as AD progressed. Based on these findings, we hypothesize that a partial deficiency of Drp1 inhibits Drp1-Aß interactions and protects Aß-induced mitochondrial and synaptic toxicities, and maintains mitochondrial dynamics and neuronal function in AD neurons. We crossed Drp1+/- mice with APP transgenic mice (Tg2576 line) and created double mutant (APPXDrp1+/-) mice. Using real-time RT-PCR and immunoblotting analyses, we measured mRNA expressions and protein levels of genes related to the mitochondrial dynamics, mitochondrial biogenesis and synapses from 6-month-old Drp1+/-, APP, APPXDrp1+/- and wild-type (WT) mice. Using biochemical methods, we also studied mitochondrial function and measured soluble Aß in brain tissues from all lines of mice in our study. Decreased mRNA expressions and protein levels of Drp1 and Fis1 (fission) and CypD (matrix) genes, and increased levels of Mfn1, Mfn2 and Opa1 (fusion), Nrf1, Nrf2, PGC1α, TFAM (biogenesis) and synaptophysin, PSD95, synapsin 1, synaptobrevin 1, neurogranin, GAP43 and synaptopodin (synaptic) were found in 6-month-old APPXDrp1+/- mice relative to APP mice. Mitochondrial functional assays revealed that mitochondrial dysfunction is reduced in APPXDrp1+/- mice relative to APP mice, suggesting that reduced Drp1enhances mitochondrial function in AD neurons. Sandwich ELISA assay revealed that soluble Aß levels were significantly reduced in APPXDrp1+/- mice relative to APP mice, indicating that reduced Drp1 decreases soluble Aß production in AD progression. These findings suggest that a partial reduction of Drp1 reduces Aß production, reduces mitochondrial dysfunction, and maintains mitochondrial dynamics, enhances mitochondrial biogenesis and synaptic activity in APP mice. These findings may have implications for the development of Drp1 based therapeutics for AD patients.

Reduced dynamin-related protein 1 protects against phosphorylated Tau-induced mitochondrial dysfunction and synaptic damage in Alzheimer's disease.

The purpose of our study was to understand the protective effects of a partial reduction of dynamin-related protein 1 (Drp1) in Alzheimer's disease (AD) progression and pathogenesis. Increasing evidence suggests that phosphorylated Tau and mitochondrial abnormalities are involved in the loss of synapses, defective axonal transport and cognitive decline, in patients with AD. In the current study, we investigated whether a partial reduction of Drp1 protect neurons from phosphorylated Tau-induced mitochondrial and synaptic toxicities in AD progression. We crossed Drp1+/− mice with Tau transgenic mice (P301L line) and created double mutant (TauXDrp1+/−) mice. Using real-time RT-PCR, immunoblotting and immunostaining analyses, we measured mRNA expressions and protein levels of genes related to the mitochondrial dynamics­Drp1 and Fis1 (fission), Mfn1, Mfn2 and Opa1 (fusion), CypD (matrix), mitochondrial biogenesis­Nrf1, Nrf2, PGC1α and TFAM and synaptic­synaptophysin, PSD95, synapsin 1, synaptobrevin 1, neurogranin, GAP43 and synaptopodin in brain tissues from 6-month-old Drp1+/−, Tau, TauXDrp1+/− and wild-type mice. Using biochemical and immunoblotting methods, mitochondrial function and phosphorylated Tau were measured. Decreased mRNA and protein levels of fission and matrix and increased levels of fusion, mitochondrial biogenesis, and synaptic genes were found in 6-month-old TauXDrp1+/− mice relative to Tau mice. Mitochondrial dysfunction was reduced in TauXDrp1+/− mice relative to Tau mice. Phosphorylated Tau found to be reduced in TauXDrp1+/− mice relative to Tau mice. These findings suggest that a partial reduction of Drp1 decreases the production of phosphorylated Tau, reduces mitochondrial dysfunction, and maintains mitochondrial dynamics, enhances mitochondrial biogenesis and synaptic activity in Tau mice. Findings of this study may have implications for the development of Drp1 based therapeutics for patients with AD and other tauopathies.

Crystal Structures of the Human Doublecortin C- and N-terminal Domains in Complex with Specific Antibodies.

Doublecortin is a microtubule-associated protein produced during neurogenesis. The protein stabilizes microtubules and stimulates their polymerization, which allows migration of immature neurons to their designated location in the brain. Mutations in the gene that impair doublecortin function and cause severe brain formation disorders are located on a tandem repeat of two doublecortin domains. The molecular mechanism of action of doublecortin is only incompletely understood. Anti-doublecortin antibodies, such as the rabbit polyclonal Abcam 18732, are widely used as neurogenesis markers. Here, we report the generation and characterization of antibodies that bind to single doublecortin domains. The antibodies were used as tools to obtain structures of both domains. Four independent crystal structures of the N-terminal domain reveal several distinct open and closed conformations of the peptide linking N- and C-terminal domains, which can be related to doublecortin function. An NMR assignment and a crystal structure in complex with a camelid antibody fragment show that the doublecortin C-terminal domain adopts the same well defined ubiquitin-like fold as the N-terminal domain, despite its reported aggregation and molten globule-like properties. The antibodies' unique domain specificity also renders them ideal research tools to better understand the role of individual domains in doublecortin function. A single chain camelid antibody fragment specific for the C-terminal doublecortin domain affected microtubule binding, whereas a monoclonal mouse antibody specific for the N-terminal domain did not. Together with steric considerations, this suggests that the microtubule-interacting doublecortin domain observed in cryo-electron micrographs is the C-terminal domain rather than the N-terminal one.

Salinization and arsenic contamination of surface water in southwest Bangladesh.

To identify the causes of salinization and arsenic contamination of surface water on an embanked island (i.e., polder) in the tidal delta plain of SW Bangladesh we collected and analyzed water samples in the dry (May) and wet (October) seasons in 2012-2013. Samples were collected from rice paddies (wet season), saltwater ponds used for brine shrimp aquaculture (dry season), freshwater ponds and tidal channels (both wet and dry season), and rainwater collectors. Continuous measurements of salinity from March 2012 to February 2013 show that tidal channel water increases from ~0.15 ppt in the wet season up to ~20 ppt in the dry season. On the polder, surface water exceeds the World Health Organization drinking water guideline of 10 µg As/L in 78% of shrimp ponds and 27% of rice paddies, raising concerns that produced shrimp and rice could have unsafe levels of As. Drinking water sources also often have unsafe As levels, with 83% of tubewell and 43% of freshwater pond samples having >10 µg As/L. Water compositions and field observations are consistent with shrimp pond water being sourced from tidal channels during the dry season, rather than the locally saline groundwater from tubewells. Irrigation water for rice paddies is also obtained from the tidal channels, but during the wet season when surface waters are fresh. Salts become concentrated in irrigation water through evaporation, with average salinity increasing from 0.43 ppt in the tidal channel source to 0.91 ppt in the rice paddies. Our observations suggest that the practice of seasonally alternating rice and shrimp farming in a field has a negligible effect on rice paddy water salinity. Also, shrimp ponds do not significantly affect the salinity of adjacent surface water bodies or subjacent groundwater because impermeable shallow surface deposits of silt and clay mostly isolate surface water bodies from each other and from the shallow groundwater aquifer. Bivariate plots of conservative element concentrations show that all surface water types lie on mixing lines between dry season tidal channel water and rainwater, i.e., all are related by varying degrees of salinization. High As concentrations in dry season tidal channel water and shrimp ponds likely result from groundwater exfiltration and upstream irrigation in the dry season. Arsenic is transferred from tidal channels to rice paddies through irrigation. Including groundwater samples from the same area (Ayers et al. in Geochem Trans 17:1-22, 2016), principal components analysis and correlation analysis reveal that salinization explains most variation in surface water compositions, whereas progressive reduction of buried surface water by dissolved organic carbon is responsible for the nonconservative behavior of S, Fe, and As and changes in Eh and alkalinity of groundwater.

Mitigation of reactive metabolite formation for a series of 3-amino-2-pyridone inhibitors of Bruton's tyrosine kinase (BTK).

Reactive metabolites have been putatively linked to many adverse drug reactions including idiosyncratic toxicities for a number of drugs with black box warnings or withdrawn from the market. Therefore, it is desirable to minimize the risk of reactive metabolite formation for lead molecules in optimization, in particular for non-life threatening chronic disease, to maximize benefit to risk ratio. This article describes our effort in addressing reactive metabolite issues for a series of 3-amino-2-pyridone inhibitors of BTK, e.g. compound 1 has a value of 459pmol/mg protein in the microsomal covalent binding assay. Parallel approaches were taken to successfully resolve the issues: establishment of a predictive screening assay with correlation association of covalent binding assay, identification of the origin of reactive metabolite formation using MS/MS analysis of HLM as well as isolation and characterization of GSH adducts. This ultimately led to the discovery of compound 7 (RN941) with significantly reduced covalent binding of 26pmol/mg protein.

Use of Artificial Intelligence and Machine Learning Algorithms with Gene Expression Profiling to Predict Recurrent Nonmuscle Invasive Urothelial Carcinoma of the Bladder.

PURPOSE: Due to the high recurrence risk of nonmuscle invasive urothelial carcinoma it is crucial to distinguish patients at high risk from those with indolent disease. In this study we used a machine learning algorithm to identify the genes in patients with nonmuscle invasive urothelial carcinoma at initial presentation that were most predictive of recurrence. We used the genes in a molecular signature to predict recurrence risk within 5 years after transurethral resection of bladder tumor. MATERIALS AND METHODS: Whole genome profiling was performed on 112 frozen nonmuscle invasive urothelial carcinoma specimens obtained at first presentation on Human WG-6 BeadChips (Illumina®). A genetic programming algorithm was applied to evolve classifier mathematical models for outcome prediction. Cross-validation based resampling and gene use frequencies were used to identify the most prognostic genes, which were combined into rules used in a voting algorithm to predict the sample target class. Key genes were validated by quantitative polymerase chain reaction. RESULTS: The classifier set included 21 genes that predicted recurrence. Quantitative polymerase chain reaction was done for these genes in a subset of 100 patients. A 5-gene combined rule incorporating a voting algorithm yielded 77% sensitivity and 85% specificity to predict recurrence in the training set, and 69% and 62%, respectively, in the test set. A singular 3-gene rule was constructed that predicted recurrence with 80% sensitivity and 90% specificity in the training set, and 71% and 67%, respectively, in the test set. CONCLUSIONS: Using primary nonmuscle invasive urothelial carcinoma from initial occurrences genetic programming identified transcripts in reproducible fashion, which were predictive of recurrence. These findings could potentially impact nonmuscle invasive urothelial carcinoma management.

Sources of salinity and arsenic in groundwater in southwest Bangladesh.

BACKGROUND: High salinity and arsenic (As) concentrations in groundwater are widespread problems in the tidal deltaplain of southwest Bangladesh. To identify the sources of dissolved salts and As, groundwater samples from the regional shallow Holocene aquifer were collected from tubewells during the dry (May) and wet (October) seasons in 2012-2013. Thirteen drill cores were logged and 27 radiocarbon ages measured on wood fragments to characterize subsurface stratigraphy. RESULTS: Drill cuttings, exposures in pits and regional studies reveal a >5 m thick surface mud cap overlying a ~30 m thick upper unit of interbedded mud and fine sand layers, and a coarser lower unit up to 60 m thick dominated by clean sands, all with significant horizontal variation in bed continuity and thickness. This thick lower unit accreted at rates of ~2 cm/year through the early Holocene, with local subsidence or compaction rates of 1-3 mm/year. Most tubewells are screened at depths of 15-52 m in sediments deposited 8000-9000 YBP. Compositions of groundwater samples from tubewells show high spatial variability, suggesting limited mixing and low and spatially variable recharge rates and flow velocities. Groundwaters are Na-Cl type and predominantly sulfate-reducing, with specific conductivity (SpC) from 3 to 29 mS/cm, high dissolved organic carbon (DOC) 11-57 mg/L and As 2-258 ug/L, and low sulfur (S) 2-33 mg/L. CONCLUSIONS: Groundwater compositions can be explained by burial of tidal channel water and subsequent reaction with dissolved organic matter, resulting in anoxia, hydrous ferric oxide (HFO) reduction, As mobilization, and sulfate (SO4) reduction and removal in the shallow aquifer. Introduction of labile organic carbon in the wet season as rice paddy fertilizer may also cause HFO reduction and As mobilization. Variable modern recharge occurred in areas where the clay cap pinches out or is breached by tidal channels, which would explain previously measured (14)C groundwater ages being less than depositional ages. Of samples collected from the shallow aquifer, Bangladesh Government guidelines are exceeded in 46 % for As and 100 % for salinity.

Drinking water insecurity: water quality and access in coastal south-western Bangladesh.

National drinking water assessments for Bangladesh do not reflect local variability, or temporal differences. This paper reports on the findings of an interdisciplinary investigation of drinking water insecurity in a rural coastal south-western Bangladesh. Drinking water quality is assessed by comparison of locally measured concentrations to national levels and water quality criteria; resident's access to potable water and their perceptions are based on local social surveys. Residents in the study area use groundwater far less than the national average; salinity and local rainwater scarcity necessitates the use of multiple water sources throughout the year. Groundwater concentrations of arsenic and specific conductivity (SpC) were greater than surface water (pond) concentrations; there was no statistically significant seasonal difference in mean concentrations in groundwater, but there was for ponds, with arsenic higher in the dry season. Average arsenic concentrations in local water drinking were 2-4 times times the national average. All of the local groundwater samples exceeded the Bangladesh guidance for SpC, although the majority of residents surveyed did not perceive their water as having a 'bad' or 'salty' taste.

Activation of the p53 pathway by small-molecule-induced MDM2 and MDMX dimerization.

Activation of p53 tumor suppressor by antagonizing its negative regulator murine double minute (MDM)2 has been considered an attractive strategy for cancer therapy and several classes of p53-MDM2 binding inhibitors have been developed. However, these compounds do not inhibit the p53-MDMX interaction, and their effectiveness can be compromised in tumors overexpressing MDMX. Here, we identify small molecules that potently block p53 binding with both MDM2 and MDMX by inhibitor-driven homo- and/or heterodimerization of MDM2 and MDMX proteins. Structural studies revealed that the inhibitors bind into and occlude the p53 pockets of MDM2 and MDMX by inducing the formation of dimeric protein complexes kept together by a dimeric small-molecule core. This mode of action effectively stabilized p53 and activated p53 signaling in cancer cells, leading to cell cycle arrest and apoptosis. Dual MDM2/MDMX antagonists restored p53 apoptotic activity in the presence of high levels of MDMX and may offer a more effective therapeutic modality for MDMX-overexpressing cancers.

Generation and external validation of a tumor-derived 5-gene prognostic signature for recurrence of lymph node-negative, invasive colorectal carcinoma.

BACKGROUND: One in 4 patients with lymph node-negative, invasive colorectal carcinoma (CRC) develops recurrent disease after undergoing curative surgery, and most die of advanced disease. Predicting which patients will develop a recurrence is a significantly growing, unmet medical need. METHODS: Archival formalin-fixed, paraffin-embedded (FFPE) primary adenocarcinoma tissues obtained at surgery were retrieved from 74 patients with CRC (15 with stage I disease and 59 with stage II disease) for Training/Test Sets. In addition, FFPE tissues were retrieved from 49 patients with stage I CRC and 215 patients with stage II colon cancer for an External Validation (EV) Set (n = 264) from 18 hospitals in 4 countries. No patients had received neoadjuvant/adjuvant therapy. Proprietary genetic programming analysis of expression profiles for 225 prespecified tumor genes was used to create a 36-month recurrence risk signature. RESULTS: Using reverse transcriptase-polymerase chain reaction, a 5-gene rule correctly classified 62 of 92 recurrent patients and 87 of 172 nonrecurrent patients in the EV Set (sensitivity, 0.67; specificity, 0.51). "High-risk" patients had a greater probability of 36-month recurrence (42%) than "low-risk" patients (26%; hazard ratio, 1.80; 95% confidence interval, 1.19-2.71; P = .007; Cox regression) independent of T-classification, the number of lymph nodes examined, histologic grade/subtype, anatomic location, age, sex, or race. The rule outperformed (P = .021) current National Comprehensive Cancer Network Guidelines (hazard ratio, 0.897). The same rule also differentiated the risk of recurrence (hazard ratio, 1.63; P = .031) in a subset of patients from the EV Set who had stage I/II colon cancer only (n = 251). CONCLUSIONS: To the authors' knowledge, the 5-gene rule (OncoDefender-CRC) is the first molecular prognostic that has been validated in both stage I CRC and stage II colon cancer. It outperforms standard clinicopathologic prognostic criteria and obviates the need to retrieve ≥12 lymph nodes for accurate prognostication. It identifies those patients most likely to develop recurrent disease within 3 years after curative surgery and, thus, those most likely to benefit from adjuvant treatment.

Antitumor activity and pharmacokinetic properties of PF-00299804, a second-generation irreversible pan-erbB receptor tyrosine kinase inhibitor.

Signaling through the erbB receptor family of tyrosine kinases contributes to the proliferation, differentiation, migration, and survival of a variety of cell types. Abnormalities in members of this receptor family have been shown to play a role in oncogenesis, thus making them attractive targets for anticancer treatments. PF-00299804 is a second-generation irreversible pan-erbB receptor tyrosine kinase inhibitor currently in phase I clinical trials. PF-00299804 is believed to irreversibly inhibit erbB tyrosine kinase activity through binding at the ATP site and covalent modification of nucleophilic cysteine residues in the catalytic domains of erbB family members. Oral administration of PF-00299804 causes significant antitumor activity, including marked tumor regressions in a variety of human tumor xenograft models that express and/or overexpress erbB family members or contain the double mutation (L858R/T790M) in erbB1 (EGFR) associated with resistance to gefitinib and erlotinib. Furthermore, PF-00299804 shows exceptional distribution to human tumor xenografts and excellent pharmacokinetic properties across species.

Pharmacologic inhibition of cyclin-dependent kinase 4/6 activity arrests proliferation in myoblasts and rhabdomyosarcoma-derived cells.

Myoblast cell cycle exit and differentiation are mediated in part by down-regulation of cyclin D1 and associated cyclin-dependent kinase (Cdk) activity. Because rhabdomyosarcoma may represent a malignant tumor composed of myoblast-like cells failing to exit the cell cycle and differentiate, we considered whether excess Cdk activity might contribute to this biology. Cyclin D-dependent Cdk4 and Cdk6 were expressed in most of a panel of six human rhabdomyosarcoma-derived cell lines. Cdk4 was expressed in 73% of alveolar and embryonal rhabdomyosarcoma tumors evaluated using a human tissue microarray. When challenged to differentiate by mitogen deprivation in vitro, mouse C2C12 myoblasts arrested in G(1) phase of the cell cycle, whereas four in the panel of rhabdomyosarcoma cell lines failed to do so. C2C12 myoblasts maintained in mitogen-rich media and exposed to a Cdk4/Cdk6 inhibitor PD 0332991 accumulated in G(1) cell cycle phase. Similar treatment of rhabdomyosarcoma cell lines caused G(1) arrest and prevented cell accumulation in vitro, and it delayed growth of rhabdomyosarcoma xenografts in vivo. Consistent with a role for Cdk4/Cdk6 activity as a regulator of myogenic differentiation, we observed that PD 0332991 exposure promoted morphologic changes and enhanced the expression of muscle-specific proteins in cultured myoblasts and in the Rh30 cell line. Our findings support the concept that pharmacologic inhibition of Cdk4/Cdk6 may represent a useful therapeutic strategy to control cell proliferation and possibly promote myogenic differentiation in rhabdomyosarcoma.

Tyrosine kinase inhibitors. 19. 6-Alkynamides of 4-anilinoquinazolines and 4-anilinopyrido[3,4-d]pyrimidines as irreversible inhibitors of the erbB family of tyrosine kinase receptors.

Structure-activity relationships for inhibition of erbB1, erbB2, and erbB4 were determined for a series of alkynamide analogues of quinazoline- and pyrido[3,4-d]pyrimidine-based compounds. The compounds were prepared by coupling the appropriate 6-aminoquinazolines or 6-aminopyrido[3,4-d]pyrimidines with alkynoic acids, using EDCI.HCl in pyridine. The compounds showed pan-erbB enzyme inhibition but were on average about 10-fold more potent against erbB1 than against erbB2 and erbB4. For cellular inhibition, the nature of the alkylating side chains was an important determinant, with 5-dialkylamino-2-pentynamide type Michael acceptors providing the highest potency. This is suggested to be due to an improved ability of the amine to participate in an autocatalysis of the Michael reaction with enzyme cysteine residues. Pyrido[3,4-d]pyrimidine analogue 39 was selected for in vivo evaluation and achieved tumor regressions at 10 mg/kg in the A431 human epidermoid carcinoma and at 40 mg/kg for the SF767 human glioblastoma and the SKOV3 human ovarian carcinoma. Complete stasis was observed at 40 mg/kg in the BXPC3 human pancreatic carcinoma as well as in the H125 human non-small-cell lung carcinoma.

The use of genetic programming in the analysis of quantitative gene expression profiles for identification of nodal status in bladder cancer.

BACKGROUND: Previous studies on bladder cancer have shown nodal involvement to be an independent indicator of prognosis and survival. This study aimed at developing an objective method for detection of nodal metastasis from molecular profiles of primary urothelial carcinoma tissues. METHODS: The study included primary bladder tumor tissues from 60 patients across different stages and 5 control tissues of normal urothelium. The entire cohort was divided into training and validation sets comprised of node positive and node negative subjects. Quantitative expression profiling was performed for a panel of 70 genes using standardized competitive RT-PCR and the expression values of the training set samples were run through an iterative machine learning process called genetic programming that employed an N-fold cross validation technique to generate classifier rules of limited complexity. These were then used in a voting algorithm to classify the validation set samples into those associated with or without nodal metastasis. RESULTS: The generated classifier rules using 70 genes demonstrated 81% accuracy on the validation set when compared to the pathological nodal status. The rules showed a strong predilection for ICAM1, MAP2K6 and KDR resulting in gene expression motifs that cumulatively suggested a pattern ICAM1 > MAP2K6 > KDR for node positive cases. Additionally, the motifs showed CDK8 to be lower relative to ICAM1, and ANXA5 to be relatively high by itself in node positive tumors. Rules generated using only ICAM1, MAP2K6 and KDR were comparably robust, with a single representative rule producing an accuracy of 90% when used by itself on the validation set, suggesting a crucial role for these genes in nodal metastasis. CONCLUSION: Our study demonstrates the use of standardized quantitative gene expression values from primary bladder tumor tissues as inputs in a genetic programming system to generate classifier rules for determining the nodal status. Our method also suggests the involvement of ICAM1, MAP2K6, KDR, CDK8 and ANXA5 in unique mathematical combinations in the progression towards nodal positivity. Further studies are needed to identify more class-specific signatures and confirm the role of these genes in the evolution of nodal metastasis in bladder cancer.

Utilizing peptide structures as keys for unlocking challenging targets.

Three-dimensional structures of protein targets have proven to be extremely valuable for modern drug design and discovery. For cases where the structure of the protein is unattainable, such as G-protein coupled receptors (GPCRs), structural information on active ligands is still useful and helpful for deciphering the geometrical and chemical features of the active site. Peptides, constructed from easy-to-form amide backbones and featuring variable side-chains, have an inherent advantage in generating rapid quantitative structure-activity relationships (QSAR). Given the fact that peptides are natural ligands for many protein targets, structural investigation of a series of related peptides, typically carried out via nuclear magnetic resonance (NMR), can result in an accurate pharmacophore model. Such a model can be used for virtual screening, and to assist design of second-generation peptidomimetics with improved properties and design of non-peptidic leads. In this article, we will review examples in which a structural approach utilizing peptide ligands was employed to obtain a better understanding of the target active site. We will focus on cases where such information supplied guidance toward the discovery of small molecule ligands.

Targeting protein-protein interactions for cancer therapy.

An increasing number of protein-protein interactions have been identified as potential intervention points for the development of anticancer agents. However, such systems have historically been considered high-risk targets due to the relatively large interaction surfaces involved in protein-protein binding. This characterization has to be reexamined as progress has been made recently in identifying small-molecule inhibitors of several protein-protein systems in oncology including the p53-MDM2 interaction. This review presents a survey of protein-protein interactions that have been identified as potential oncology targets and evaluates their attractiveness in terms of drug discovery. The analysis focuses primarily on the structural characteristics of the participating binding sites, particularly the dimensions of the sites. Known ligands are also examined, especially with regard to their druglikeness.

Plasma vascular endothelial growth factor and interleukin-8 as biomarkers of antitumor efficacy of a prototypical erbB family tyrosine kinase inhibitor.

CI-1033 (N-[4-[N-(3-chloro-4-fluorophenyl)amino-7-[3-(4-morpholynyl)propoxy]quinazolin-6-yl]acrylamide, PD 0183805-mesylate salt) was identified as a potent, selective inhibitor of erbB family tyrosine kinases, which are overexpressed in a number of solid tumors and have been shown to be involved in tumor progression. Because objective response of clinical patients to erbB-targeted therapies like CI-1033 has been observed only in a subset of cancer patients that exhibit the intended molecular targets, much emphasis has been placed on the identification of biomarkers of antitumor efficacy. Vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) were considered as potential biomarkers for CI-1033 due to ease of detection in patient plasma and showed roles in angiogenesis and cancer progression and positive regulation by the erbB receptor family. In the present studies, mice bearing established xenografts (A431 epidermoid carcinoma, H125 non-small cell lung carcinoma, SF767 glioblastoma, and MDA-MB-468 mammary carcinoma) were treated with efficacious and subefficacious doses of CI-1033, and plasma levels and xenograft gene expression of VEGF and IL-8 were evaluated. Oral administration of CI-1033 to tumor-bearing mice at efficacious doses resulted in markedly decreased levels of VEGF and/or IL-8 plasma levels and tumor mRNA levels relative to vehicle-treated control mice in xenograft models that exhibited evaluable levels of these markers. In contrast, subefficacious doses of CI-1033 did not significantly affect VEGF or IL-8 levels in any of the xenograft models. These studies indicate that plasma VEGF and IL-8 may have use as biomarkers of antitumor efficacy for epidermal growth factor receptor/erbB-targeted therapies such as CI-1033 and suggest that further clinical study of these markers in cancer patients are warranted.

Differential sensitivity of cancer cells to inhibitors of the epidermal growth factor receptor family.

Clinical responses to the HER1 (EGF receptor) inhibitors and HER2/neu/ErbB2 inhibitors correlate with high levels of receptor expression. However, a significant subset of patients with high receptor levels appear to be refractory to treatment. We have observed similar results in the 60 cell lines of the NCI Anti-Cancer Drug Screen using a panel of 11 selective HER1 inhibitors. As expected, low HER1-expressing cell lines were insensitive to HER1 inhibitors. In cell lines with high HER1 expression, low concentrations of HER1 inhibitors potently inhibit both HER1 phosphorylation and the mitogen-activated protein kinase (MAPK) pathway. However, this inhibition did not always correlate with cellular arrest. High HER1-expressing cell lines can be subdivided into two groups based on their sensitivity to HER1 inhibitors. In the sensitive group, receptor and growth inhibition was concordant and occurred at sub-micromolar concentrations of HER1 inhibitors. In the insensitive group, receptor inhibition occurred at a low concentration (< 1 microM) but concentrations that were ten times or higher were required for growth inhibition. Also, neither induction of p21 and cyclin D1 nor p53 status could explain the difference between sensitive and insensitive cells. Although EGF activated the MAPK pathway in all cell lines, only drug-sensitive cell lines responded to EGF (accelerated entry from G1 to S) and to HER1 inhibitors (G1 arrest) by changes in cell cycling. Furthermore, an EGF-dependent immortalized mammary epithelial cell line was extremely sensitive to a panel of HER1 inhibitors. We infer that independence from mitogen-mediated signaling confers insensitivity to HER1 inhibitors in a large subset of cancer cell lines.

Development of E3-substrate (MDM2-p53)-binding inhibitors: structural aspects.

Inhibition of E3 ligase-substrate binding is the most direct approach for blocking protein ubiquitylation and degradation. However, protein-protein interactions have proven to be difficult targets for discovery of small molecules that bind at the interface and modulate protein activity in a selective manner. Recently, we developed the first potent and selective small-molecule inhibitors of the binding between MDM2 E3 ligase and its substrate p53 (Vassilev et al., 2004). This process was aided significantly by the acquisition and use of structural information. We describe herein how such information was obtained and used at various stages in the program. These applications included assessment of MDM2 as a target, evaluation of hits from high-throughput screening and the selection of lead molecules, and analysis of binding strategies used by the inhibitors as a basis for guiding studies of similar systems. These tools are likely to be useful in any attempt to find and develop druglike compounds that modulate the function of a protein-protein interaction.