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.

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.

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.

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.

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.

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.

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.

Garrison Institute on Aging: A New Hope for Elderly Individuals and Patients with Alzheimer's Disease.

The Garrison Institute on Aging (GIA) is an established institute within Texas Tech University Health Sciences Center, whose mission is to promote healthy aging through cutting-edge research on Alzheimer's disease (AD) and other diseases of aging through innovative educational opportunities for students, clinicians, researchers, health care professionals, and the public. The GIA has multiple programs, including both research and education on healthy aging and AD, community outreach, caregiving, the Retired Senior Volunteer Program, Healthy Lubbock, the GIA Brain Bank, healthy aging seminars, research seminars, and collaborations and scholarships. The GIA programs connect basic and clinical researchers and health care professionals, and provide a unique environment to help our growing elderly population and patients with AD and their families.

Targeting protein-protein interactions for drug discovery.

Protein-protein interactions are associated with key activities and pathways in the cell, and in that regard are promising targets for drug discovery. However, in terms of small molecule drugs, this promise has not been realized. The physical nature of many protein-protein interaction surfaces renders them unable to support binding of small drug-like molecules. In addition, there are other unique hurdles presented by this class that make the drug development process difficult and risky. Nevertheless, success stories have begun to steadily appear in this field. These experiences are starting to provide general strategies and tools to help overcome the problems inherent in pursuing protein-protein interaction targets. These lessons should improve the rate of success as these systems are pursued in the future.

Structure-Based Design and Synthesis of Potent Cyclic Peptides Inhibiting the YAP-TEAD Protein-Protein Interaction.

The YAP-TEAD protein-protein interaction (PPI) mediates the oncogenic function of YAP, and inhibitors of this PPI have potential usage in treatment of YAP-involved cancers. Here we report the design and synthesis of potent cyclic peptide inhibitors of the YAP-TEAD interaction. A truncation study of YAP interface 3 peptide identified YAP(84-100) as a weak peptide inhibitor (IC50 = 37 µM), and an alanine scan revealed a beneficial mutation, D94A. Subsequent replacement of a native cation-π interaction with an optimized disulfide bridge for conformational constraint and synergistic effect between macrocyclization and modification at positions 91 and 93 greatly boosted inhibitory activity. Peptide 17 was identified with an IC50 of 25 nM, and the binding affinity (K d = 15 nM) of this 17mer peptide to TEAD1 proved to be stronger than YAP(50-171) (K d = 40 nM).

Design of libraries targeting protein-protein interfaces.

TARGETING PPIS: A novel strategy for designing libraries targeting protein-protein interfaces enabled us to identify diverse chemical entry points to interact with therapeutic targets for which conventional screening libraries delivered no or only few hit structures. The concept was experimentally validated by early hit evaluation in biochemical screens and early ADMET profiling.

Deconstruction of a nutlin: dissecting the binding determinants of a potent protein-protein interaction inhibitor.

Protein-protein interaction (PPI) systems represent a rich potential source of targets for drug discovery, but historically have proven to be difficult, particularly in the lead identification stage. Application of the fragment-based approach may help toward success with this target class. To provide an example toward understanding the potential issues associated with such an application, we have deconstructed one of the best established protein-protein inhibitors, the Nutlin series that inhibits the interaction between MDM2 and p53, into fragments, and surveyed the resulting binding properties using heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR), surface plasmon resonance (SPR), and X-ray crystallography. We report the relative contributions toward binding affinity for each of the key substituents of the Nutlin molecule and show that this series could hypothetically have been discovered via a fragment approach. We find that the smallest fragment of Nutlin that retains binding accesses two subpockets of MDM2 and has a molecular weight at the high end of the range that normally defines fragments.

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.

Small-molecule inhibitors of protein-protein interactions: how to mimic a protein partner.

This systematic review describes successful examples of small-molecule inhibitors of protein-protein interactions, and compares their binding strategies to those employed by the natural protein partners. It extends and updates an earlier survey of this type (Fry DC, Curr Prot Pep Sci 2008; 9: 240-7). From analysis of these systems, common themes and lessons are presented that may assist future drug discovery efforts involving targets in this class. One encouraging finding is that a wide scope appears to be allowed at these sites in terms of binding strategies and chemotypes, which suggests that the outlook for finding small-molecule protein-protein inhibitors is favorable.

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.

Druggability assessment of protein-protein interfaces.

Recent success stories concerning the targeting of protein-protein interactions (PPIs) have led to an increased focus on this challenging target class for drug discovery. This article explores various avenues to assess the druggability of PPIs and describes a druggability decision flow chart, which can be applied to any PPI target. This flow chart not only covers small molecules but also peptidomimetics, peptides and conformationally restricted peptides as potential modalities for targeting PPIs. Additionally, a retrospective analysis of PPI druggability using various computational tools is summarized. The application of a systematic approach as presented in this paper will increase confidence that modulators (e.g., small organic molecules or peptides) can ultimately be identified for a particular target before a decision is made to commit significant discovery resources.

Phase I study of bryostatin 1, a protein kinase C modulator, preceding cisplatin in patients with refractory non-hematologic tumors.

PURPOSE: Preclinical data suggested that bryostatin-1 (bryo) could potentiate the cytotoxicity of cisplatin when given prior to this drug. We designed a phase I study to achieve tolerable doses and schedules of bryo and cisplatin in combination and in this sequence. METHODS: Patients with non-hematologic malignancies received bryo followed by cisplatin in several schedules. Bryo was given as an 1 and a 24 h continuous infusion, while cisplatin was always given over 1 h at 50 and 75 mg/m(2); the combined regimen was repeated on an every 3-week and later on an every 2-week schedule. Bryo doses were escalated until recommended phase II doses were defined for each schedule. Patients were evaluated with computerized tomography every 2 cycles. RESULTS: Fifty-three patients were entered. In an every 2-week schedule, the 1-h infusion of bryo became limited by myalgia that was clearly cumulative. With cisplatin 50 mg/m(2) its recommended phase II dose was 30 microg/m(2). In the 3-week schedule, dose-limiting toxicities were mostly related to cisplatin effects while myalgias were tolerable. Pharmacokinetics unfortunately proved to be unreliable due to bryo's erratic extraction. Consistent inhibition of PKC isoform eta (eta) in peripheral blood mononuclear cells was observed following bryo. CONCLUSIONS: Bryo can be safely administered with cisplatin with minimal toxicity; however, only four patients achieved an objective response. Modulation of cisplatin cytotoxicity by bryo awaits further insight into the molecular pathways involved.