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Inhibitors of the mTOR kinase are in clinical trials for the treatment of cancer; here, mutations in mTOR that can lead to drug resistance are investigated and the results are used to design a new class of mTOR inhibitors that can overcome this resistance. Bivalent mTOR inhibitors counter tumour resistance Inhibitors of the mTOR kinase are in clinical trials for the treatment of cancer. Here Kevan Shokat and colleagues investigate mutations in mTOR that can lead to drug resistance, and identify pre-existing hyperactive kinase domain mutants in cancer patients unresponsive to mTOR treatment. They design a new class of mTOR inhibitor that can overcome this resistance by binding to two sites on the kinase simultaneously and inhibiting signalling and tumour growth in mice. Precision medicines exert selective pressure on tumour cells that leads to the preferential growth of resistant subpopulations, necessitating the development of next-generation therapies to treat the evolving cancer. The PIK3CA–AKT–mTOR pathway is one of the most commonly activated pathways in human cancers 1 , which has led to the development of small-molecule inhibitors that target various nodes in the pathway. Among these agents, first-generation mTOR inhibitors (rapalogs) have caused responses in ‘ N -of-1’ cases, and second-generation mTOR kinase inhibitors (TORKi) are currently in clinical trials 2 , 3 , 4 . Here we sought to delineate the likely resistance mechanisms to existing mTOR inhibitors in human cell lines, as a guide for next-generation therapies. The mechanism of resistance to the TORKi was unusual in that intrinsic kinase activity of mTOR was increased, rather than a direct active-site mutation interfering with drug binding. Indeed, identical drug-resistant mutations have been also identified in drug-naive patients, suggesting that tumours with activating MTOR mutations will be intrinsically resistant to second-generation mTOR inhibitors. We report the development of a new class of mTOR inhibitors that overcomes resistance to existing first- and second-generation inhibitors. The third-generation mTOR inhibitor exploits the unique juxtaposition of two drug-binding pockets to create a bivalent interaction that allows inhibition of these resistant mutants.

High-throughput screening identified a small-molecule compound that targets the active conformation of HER2 and is effective against growth-factor-mediated drug resistance. The heterodimeric receptor tyrosine kinase complex formed by HER2 and HER3 can act as an oncogenic driver and is also responsible for rescuing a large number of cancers from a diverse set of targeted therapies. Inhibitors of these proteins, particularly HER2, have dramatically improved patient outcomes in the clinic, but recent studies have demonstrated that stimulating the heterodimeric complex, either via growth factors or by increasing the concentrations of HER2 and HER3 at the membrane, significantly diminishes the activity of the inhibitors. To identify an inhibitor of the active HER2–HER3 oncogenic complex, we developed a panel of Ba/F3 cell lines suitable for ultra-high-throughput screening. Medicinal chemistry on the hit scaffold resulted in a previously uncharacterized inhibitor that acts through preferential inhibition of the active state of HER2 and, as a result, is able to overcome cellular mechanisms of resistance such as growth factors or mutations that stabilize the active form of HER2.

Summary PAC-1 is a preferential small molecule activator of procaspase-3 and has potential to become a novel and effective anticancer agent. The rational development of PAC-1 for translational oncologic applications would be advanced by coupling relevant in vitro cytotoxicity studies with pharmacokinetic investigations conducted in large mammalian models possessing similar metabolism and physiology as people. In the present study, we investigated whether concentrations and exposure durations of PAC-1 that induce cytotoxicity in lymphoma cell lines in vitro can be achievable in healthy dogs through a constant rate infusion (CRI) intravenous delivery strategy. Time- and dose-dependent procaspase-3 activation by PAC-1 with subsequent cytotoxicity was determined in a panel of B-cell lymphoma cells in vitro. The pharmacokinetics of PAC-1 administered orally or intravenously was studied in 6 healthy dogs using a crossover design. The feasibility of maintaining steady state plasma concentration of PAC-1 for 24 or 48 h that paralleled in vitro cytotoxic concentrations was investigated in 4 healthy dogs. In vitro, PAC-1 induced apoptosis in lymphoma cell lines in a time- and dose-dependent manner. The oral bioavailability of PAC-1 was relatively low and highly variable (17.8 ± 9.5%). The achievement and maintenance of predicted PAC-1 cytotoxic concentrations in normal dogs was safely attained via intravenous CRI lasting for 24 or 48 h in duration. Using the dog as a large mammalian model, PAC-1 can be safely administered as an intravenous CRI while achieving predicted in vitro cytotoxic concentrations.

Targeted inhibitors of driving oncogenes impose a selective pressure that benefits those cells who can most quickly and efficiently bypass the insult. While much attention, particularly in the kinase inhibitor field, has focused on the selective outgrowth of cells harboring mutations that directly impact the inhibitors ability to bind a much more rapid innate resistance mechanism often exists that it is revealed by the presence of the inhibitor. In chapter 1 we found that growth factor driven resistance to current clinical HER2 inhibitors was due to the inability of the inhibitors to directly target the active state of the kinase. We used this information to design a screen for an inhibitor of the active HER2/HER3 complex resulting in the identification of one hit scaffold. Optimization of our small molecule hit resulted in a HER2 inhibitor that was insensitive the presence of growth factor and also capable of targeting the mutationally activated form of HER2. In chapter 2 we used the existing known biology of K-Ras’s ability to bypass first generation farnesyltransferase inhibitors to design an inhibitor that would simultaneously block K-Ras cellular localization and its ability to bypass inhibition. The resulting inhibitor was found to block K-Ras localization in cells, and when combined with statins, was found to inhibit oncogenic K-Ras signaling and proliferation.

PAC-1 is a preferential small molecule activator of procaspase-3 and has potential to become a novel and effective anticancer agent. The rational development of PAC-1 for translational oncologic applications would be advanced by coupling relevant in vitro cytotoxicity studies with pharmacokinetic investigations conducted in large mammalian models possessing similar metabolism and physiology as people. In the present study, we investigated whether concentrations and exposure durations of PAC-1 that induce cytotoxicity in lymphoma cell lines in vitro can be achievable in healthy dogs through a constant rate infusion (CRI) intravenous delivery strategy. Time- and dose-dependent procaspase-3 activation by PAC-1 with subsequent cytotoxicity was determined in a panel of B-cell lymphoma cells in vitro. The pharmacokinetics of PAC-1 administered orally or intravenously was studied in 6 healthy dogs using a crossover design. The feasibility of maintaining steady state plasma concentration of PAC-1 for 24 or 48 hours that paralleled in vitro cytotoxic concentrations was investigated in 4 healthy dogs. In vitro, PAC-1 induced apoptosis in lymphoma cell lines in a time- and dose-dependent manner. The oral bioavailability of PAC-1 was relatively low and highly variable (17.8 ± 9.5%). The achievement and maintenance of predicted PAC-1 cytotoxic concentrations in normal dogs was safely attained via intravenous CRI lasting for 24 or 48 hours in duration. Using the dog as a large mammalian model, PAC-1 can be safely administered as an intravenous CRI while achieving predicted in vitro cytotoxic concentrations.

Bacteria in the 16S rRNA clade SAR86 are among the most abundant uncultivated constituents of microbial assemblages in the surface ocean for which little genomic information is currently available. Bioinformatic techniques were used to assemble two nearly complete genomes from marine metagenomes and single-cell sequencing provided two more partial genomes. Recruitment of metagenomic data shows that these SAR86 genomes substantially increase our knowledge of non-photosynthetic bacteria in the surface ocean. Phylogenomic analyses establish SAR86 as a basal and divergent lineage of γ-proteobacteria, and the individual genomes display a temperature-dependent distribution. Modestly sized at 1.25–1.7 Mbp, the SAR86 genomes lack several pathways for amino-acid and vitamin synthesis as well as sulfate reduction, trends commonly observed in other abundant marine microbes. SAR86 appears to be an aerobic chemoheterotroph with the potential for proteorhodopsin-based ATP generation, though the apparent lack of a retinal biosynthesis pathway may require it to scavenge exogenously-derived pigments to utilize proteorhodopsin. The genomes contain an expanded capacity for the degradation of lipids and carbohydrates acquired using a wealth of tonB-dependent outer membrane receptors. Like the abundant planktonic marine bacterial clade SAR11, SAR86 exhibits metabolic streamlining, but also a distinct carbon compound specialization, possibly avoiding competition.

Purpose To externally validate the Christodouleas risk model incorporating pathological tumor stage, lymph node (LN) count and soft tissue surgical margin (STSM) and stratifying patients who develop locoregional recurrence (LR) after radical cystectomy (RC) for urothelial carcinoma of the bladder (UCB). In addition, we aimed to generate a new model including established clinicopathological features that were absent in the Christodouleas risk model. Methods Prospectively assessed multicenter data from 565 patients undergoing RC for UCB in 2011 qualified for final analysis. For the purpose of external validation, risk group stratification according to Christodouleas was performed. Competing-risk models were calculated to compare the cumulative incidences of LR after RC. Results After a median follow-up of 25 months (interquartile range 19–29), the LR-rate was 11.5 %. The Christodouleas model showed a predictive accuracy of 83.2 % in our cohort. In multivariable competing-risk analysis, tumor stage ≥pT3 (HR 4.32, p  < 0.001), positive STSM (HR 2.93, p  = 0.005), lymphovascular invasion (HR 3.41, p  < 0.001), the number of removed LNs <10 (HR 2.62, p  < 0.001) and the administration of adjuvant chemotherapy (HR 0.40, p  = 0.008) independently predicted the LR-rate. The resulting risk groups revealed significant differences in LR-rates after 24 months with 4.8 % for low-risk patients, 14.7 % for intermediate-risk patients and 38.9 % for high-risk patients ( p  < 0.001 for all), with a predictive accuracy of 85.6 %, respectively. Conclusions The Christodouleas risk model has been successfully externally validated in the present prospective series. However, this analysis finds that overall model performance may be improved by incorporating lymphovascular invasion. After external validation of the newly proposed risk model, it may be used to identify patients who benefit from an adjuvant therapy and suit for inclusion in clinical trials.

Aim: Insects feeding on seeds and fruits represent interesting study systems, potentially able to lower the fitness of their host plants. In addition to true seed eaters, a suite of insects feed on the fleshy parts of fruits. We examined the likelihood of community convergence in whole insect assemblages attacking seeds/fruits in three tropical rain forests. Location: Three ForestGEO permanent forest plots within different biogeographical regions: Barro Colorado Island (Panama), Khao Chong (Thailand) and Wanang (Papua New Guinea). Methods: We surveyed 1,186 plant species and reared 1.1 ton of seeds/fruits that yielded 80,600 insects representing at least 1,678 species. We assigned seeds/fruits to predation syndromes on the basis of plant traits relevant to insects, seed/fruit appearance and mesocarp thickness. Results: We observed large differences in insect faunal composition, species richness and guild structure between our three study sites. We hypothesize that the high species richness of insect feeding on seeds/fruits in Panama may result from a conjunction of low plant species richness and high availability of dry fruits. Insect assemblages were weakly influenced by seed predation syndromes, both at the local and regional scale, and the effect of host phylogeny varied also among sites. At the driest site (Panama), the probability of seeds of a plant species being attacked depended more on seed availability than on the measured seed traits of that plant species. However, when seeds were attacked, plant traits shaping insect assemblages were difficult to identify and not related to seed availability. Main conclusions: We observed only weak evidence of community convergence at the intercontinental scale among these assemblages. Our study suggests that seed eaters may be most commonly associated with dry fruits at relatively dry tropical sites where fleshy fruits may be less prevalent.

Major tropical rivers have been suggested to be important dispersal barriers that increase the beta diversity of animal communities in lowland rain forests. We tested this hypothesis using assemblages of frogs in the floodplains of the Sepik River, a major river system in Papua New Guinea. We surveyed frogs at five sites within a continuous 150 × 500-km area of lowland rain forest bisected by the Sepik, using standardized visual and auditory survey techniques. We documented 769 frogs from 44 species. The similarity in species composition decreased with logarithm of geographical distance between the sites, which ranged from 82 to 465 km. The similarity decay did not depend on whether or not the compared sites were separated by the Sepik River or whether the species were aquatic or terrestrial breeders. Likewise, a DCA ordination of frog assemblages did not show separation of sites by the river as a significant factor explaining their composition. Our results suggest that even major rivers, such as the Sepik, may not act as dispersal barriers. Rivers may not limit the distribution of frogs and therefore have a limited effect on determining frog species abundance and assemblage structure in rain forests.

We propose a new classification of rain forest plants into eight fruit syndromes, based on fruit morphology and other traits relevant to fruit-feeding insects. This classification is compared with other systems based on plant morphology or traits relevant to vertebrate fruit dispersers. Our syndromes are based on fruits sampled from 1,192 plant species at three Forest Global Earth Observatory plots: Barro Colorado Island (Panama), Khao Chong (Thailand), and Wanang (Papua New Guinea). The three plots differed widely in fruit syndrome composition. Plant species with fleshy, indehiscent fruits containing multiple seeds were important at all three sites. However, in Panama, a high proportion of species had dry fruits, while in New Guinea and Thailand, species with fleshy drupes and thin mesocarps were dominant. Species with dry, winged seeds that do not develop as capsules were important in Thailand, reflecting the local importance of Dipterocarpaceae. These differences can also determine differences among frugivorous insect communities. Fruit syndromes and colors were phylogenetically flexible traits at the scale studied, as only three of the eight seed syndromes, and one of the 10 colors, showed significant phylogenetic clustering at either genus or family levels. Plant phylogeny was, however, the most important factor explaining differences in overall fruit syndrome composition among individual plant families or genera across the three study sites.