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Inferences made from molecular data support regional stock assessment goals by providing insights into the genetic population dynamics of enigmatic species. Population genomics metrics, such as genetic diversity and population connectivity, serve as useful proxies for species health and stability. Sleeper sharks (genus Somniosus) are ecologically important deep-sea predators, estimated to reach ages of 250 to 300 yr and taking decades to reach sexual maturity. The subgenus Somniosus (Somniosus) is comprised of 3 species: S. pacificus, S. microcephalus, and S. antarcticus. Given the life history strategy of somniosids, they are vulnerable to overfishing and population declines. Further, data to assess the stocks of these species are limited. To address this deficiency, we used the reduced representation library method Restriction-site Associated DNA sequencing (RADseq) to conduct phylogenomic and population genomics analyses, providing novel information for use in stock assessments. Our results strongly support the species status of S. microcephalus (N = 79), but recover S. antarcticus (N = 2) intermixed within the S. pacificus (N = 170) clade. Population genomics analyses reveal genetic homogeneity within S. pacificus and S. microcephalus, and estimates of effective population size were in the hundreds for both species. Kinship analysis identified 2 first-degree relative pairs within our dataset (1 within each species). Our results contribute new information for stock assessments of these uniquely long-lived species by providing the strongest molecular evidence to date for the synonymization of S. antarcticus and S. pacificus, as well as estimating population genomic metrics for each supported species within the Somniosus (Somniosus) subgenus.

Diabrotica undecimpunctata is a multivoltine polyphagous beetle species that has long been documented as a significant agricultural pest throughout its native range in North America. This beetle can vector bacterial and viral plant pathogens that result in major losses to crops such as cucumber and soybean. Many countries outside the Americas treat D. undecimpunctata as a species of quarantine importance, while in the USA only the subspecies D. u. duodecimnotata is subject to quarantine, to prevent introduction from Mexico. Identification of D. undecimpunctata on the basis of morphology alone can be complicated given the use of conflicting characters in the description of some subspecific taxa. To better understand relationships among D. undecimpunctata subspecies and other related species, we sequenced mitochondrial cytochrome oxidase 1 (CO1) and nuclear internal transcribed spacer 2 (ITS2) DNA from individuals in different subspecific taxa and across different parts of the species range using museum samples and interceptions. When our data were combined with publicly available Diabrotica data, no pattern of divergence consistent with the currently recognized subspecific designations was found. In addition, we compared phylogenetic patterns in CO1 data from the congener D. virgifera to demonstrate the utility of mitochondrial data in resolving subspecies. From the CO1 data, a diagnostic real-time PCR assay was developed that could successfully identify all haplotypes within the large D. undecimpunctata clade for use in surveys and identification at ports of entry. These findings underscore the need to resolve molecular and morphological datasets into cogent, lineage-based groupings. Such efforts will provide an evolutionary context for the study of agriculturally important attributes of Diabrotica such as host preferences, xenobiotic metabolism, and natural and anthropogenic patterns of dispersal.

The proteasome is a multi-subunit proteolytic complex that functions to degrade normal proteins for physiological regulation and to eliminate abnormal proteins for cellular protection. Generally, the proteasome targets substrate proteins that are marked by attachment of multiple ubiquitin molecules. In various types of cells in an organism, damage to proteins occurs both from internal sources such as reactive oxygen species and from external ones such as UV radiation from the sun. The proteasome functions to protect the cells by degrading damaged proteins. With ageing, however, the capacity of the proteasome to degrade damaged proteins is reduced as indicated by evidence gathered by many studies. Studies on ageing in muscle, skin, and brain show that with age catalytic activity of the proteasome is decreased and the expression of proteasome subunits is altered. Age-related accumulation of damaged or misfolded proteins causes further reduction of proteasome activity. Abnormal proteins also accumulate as a result of age-related neurodegenerative diseases. Deficits in proteasome activity might be responsible for accumulation of protein aggregates and thus contribute to the pathology. Results from several studies suggest a link between the proteasome and longevity. This chapter reviews the various ways in which the proteasome is associated with the ageing process and examines evidence gathered from investigations on cultured cells, model organisms, and humans.

PRMT5 and its substrate adaptor proteins (SAPs), pICln and Riok1, are synthetic lethal dependencies in MTAP-deleted cancer cells. SAPs share a conserved PRMT5 binding motif (PBM) which mediates binding to a surface of PRMT5 distal to the catalytic site. This interaction is required for methylation of several PRMT5 substrates, including histone and spliceosome complexes. We screened for small molecule inhibitors of the PRMT5-PBM interaction and validated a compound series which binds to the PRMT5-PBM interface and directly inhibits binding of SAPs. Mode of action studies revealed the formation of a covalent bond between a halogenated pyridazinone group and cysteine 278 of PRMT5. Optimization of the starting hit produced a lead compound, BRD0639, which engages the target in cells, disrupts PRMT5-RIOK1 complexes, and reduces substrate methylation. BRD0639 is a first-in-class PBM-competitive inhibitor that can support studies of PBM-dependent PRMT5 activities and the development of novel PRMT5 inhibitors that selectively target these functions.

Drilling a minimally invasive access to the inner ear is a demanding task in which a computer-assisted surgical system can support the surgeon. Herein, we describe the design of a new micro-stereotactic targeting system dedicated to cochlear implant (CI) surgery and its experimental evaluation in an ex vivo study.The proposed system consists of a reusable, bone-anchored reference frame, and a patient-specific drilling jig on top of it. Individualization of the jig is simplified to a single counterbored hole drilled out of a blank. For accurate counterboring, the setup includes a manufacturing device for individual positioning of the blank. The system was tested in a preclinical setting using twelve human cadaver donors. Cone beam computed tomograph (CBCT) scans were obtained and a drilling trajectory was planned pointing towards the basal part of the cochlea. The surgical drill was moved forward manually and slowly while the jig constrained the drill along the predetermined path.Drilling could be performed with preservation of facial nerve in all specimens. The mean error caused by the system at the target point in front of the cochlea was 0.30 mm ± 0.11 mm including an inaccuracy of 0.09 mm ± 0.03 mm for counterboring the guiding aperture into the jig.Feasibility of the proposed system to perform a minimally invasive posterior tympanotomy approach was shown successfully in all specimens.First evaluation of the new system in a comprehensive ex vivo study demonstrating sufficient accuracy and the feasibility of the whole concept.

The moth species Phthorimaea absoluta (Meyrick) (formerly Tuta absoluta) is serious threat to tomato and other Solanaceous crops worldwide and is invasive throughout Europe, Asia, and Africa. While P. absoluta has not yet been found in the U.S. recent detections in the Caribbean have raised concerns that the species could be introduced to mainland North America. To improve detection capacity, a droplet digital PCR (ddPCR) assay was developed that employs a nondestructive bulk DNA extraction method able to detect one P. absoluta sample among 200 nontargets. Such high-throughput and sensitive molecular assays are essential to preventing introductions through early detection and response. This assay can also be used in areas where P. absoluta is established to monitor outbreaks and track migratory patterns.

The tomato leafminer, Tuta absoluta (Meyrick), is a highly destructive pest of tomatoes, causing damage to leaves, stalks, buds, and fruits. Native to South America, T. absoluta is now found throughout Europe, South Asia, Africa, parts of Central America, and the Caribbean. Adults are small, with a wingspan of approximately one cm and lack distinctive markings, making morphological identification difficult. Larvae are also difficult to identify and resemble those of many other gelechiids. Due to the extensive time spent and expertise required for morphological identification, and the imminent threat to the North American tomato crop, we have developed a rapid molecular test for discriminating individual specimens of T. absoluta using a probe-based real-time polymerase chain reaction (PCR) assay. The assay is able to quickly distinguish T. absoluta from similar-sized moth specimens that are attracted to T. absoluta pheromone lures in the United States and is also able to identify larvae of T. absoluta. Decreased identification time for this critical pest will lead to more rapid identification at ports of entry and allow for more efficient trap screening for domestic monitoring programs.

Simple Summary Invasive species are a constant threat to agriculture throughout the world against which early detection is one of the primary defenses. The Old World bollworm is one of the most important invasive agricultural pests in the world. While historically absent from the Americas, this species was first found in South America in 2013 and poses an ongoing threat of spreading into North America. Surveys are conducted each year, which result in hundreds or thousands of traps that must be screened for this species. Unfortunately, the most common non-target is the native corn earworm, which is nearly identical morphologically to the Old World bollworm and cannot be easily separated. Molecular methods have been developed to screen these trap samples, but the required equipment is expensive and not commonly available. This study details improvements to current molecular methods that will allow for screening of bulk trap samples using standard laboratory instruments and protocols. The ability to perform these methods in nearly any molecular biology lab will greatly enhance our ability to detect and exclude this important pest. Abstract Helicoverpa armigera (Hübner) is one of the most important agricultural pests in the world. This historically Old World species was first reported in Brazil in 2013 and has since spread throughout much of South America and into the Caribbean. Throughout North America, H. armigera surveys are ongoing to detect any incursions. Each trap is capable of capturing hundreds of native Helicoverpa zea (Boddie). The two species cannot be separated without genitalic dissection or molecular methods. A ddPCR assay is currently used to screen large trap samples, but this equipment is relatively uncommon and expensive. Here, we optimized a newly designed assay for accurate and repeatable detection of H. armigera in bulk samples across both ddPCR and less costly, and more common, real-time PCR methods. Improvements over previously designed assays were sought through multiple means. Our results suggest bulk real-time PCR assays can be improved through changes in DNA extraction and purification, so that real-time PCR can be substituted for ddPCR in screening projects. While ddPCR remains a more sensitive method for detection of H. armigera in bulk samples, the improvements in assay design, DNA extraction, and purification presented here also enhance assay performance over previous protocols.

The Gulf of Mexico pink shrimp, Farfantepenaeus duorarum, supports large fisheries in the United States and Mexico, with nearly 7,000 tons harvested from the region in 2016. Given the commercial importance of this species, management is critical: in 1997, the southern Gulf of Mexico pink shrimp fishery was declared collapsed and mitigation strategies went into effect, with recovery efforts lasting over a decade. Fisheries management can be informed and improved through a better understanding of how factors associated with early life history impact genetic diversity and population structure in the recruited population. Farfantepenaeus duorarum are short-lived, but highly fecund, and display high variability in recruitment patterns. To date, modeling the impacts of ecological, physical, and behavioral factors on juvenile settlement has focused on recruitment of larval individuals of F. duorarum to nursery grounds in Florida Bay. Here, we articulate testable hypotheses stemming from a recent model of larval transport and evaluate support for each with a population genomics approach, generating reduced representation library sequencing data for F. duorarum collected from seven regions around the Florida Peninsula. Our research represents the first and most molecular data-rich study of population structure in F. duorarum in the Gulf and reveals evidence of a differentiated population in the Dry Tortugas. Our approach largely validates a model of larval transport, allowing us to make management-informative inferences about the impacts of spawning location and recruitment patterns on intraspecific genetic diversity. Such inferences improve our understanding of the roles of non-genetic factors in generating and maintaining genetic diversity in a commercially important penaeid shrimp species.

Bark beetles in the family Curculionidae present a growing hazard to forests worldwide. Like native bark beetles, introduced exotic species can pose a serious threat to North American forests. Ips typographus (Boerner) and Ips sexdentatus (Linnaeus), both native to Europe, are two such pests that have caused widespread forest loss in their native ranges. International trade has led to increased interceptions of Scolytine beetles at ports of entry to the United States. Most intercepted individuals are not identified to species due to lack of expert identifiers, poor specimen quality, or incomplete taxonomy. These same problems affect identification for domestic surveys. Therefore, development of molecular methods for identification of potentially invasive Ips species is essential. Because of the need to scrutinize large numbers of beetles in an efficient manner, we describe a duplex droplet digital PCR (ddPCR) assay to identify I. typographus and I. sexdentatus simultaneously in bulk trap samples containing 500 Scolytinae specimens using a scalable, two-step DNA extraction. This ddPCR method is highly effective for processing the entire contents of beetle traps and identifying these potentially invasive species in a timely and definitive manner. We also describe a nondestructive DNA extraction technique that preserves specimens for morphological identification.

The Old World bollworm, Helicoverpa armigera (Hübner), is one of the most destructive agricultural pests worldwide. It was first recorded in Brazil in 2013, yet despite this recent introduction, H. armigera has spread throughout much of Latin America. Where H. armigera has become established, it is displacing or hybridizing with the congeneric New World pest Helicoverpa zea. In addition to the adaptive qualities that make H. armigera a megapest, such as broad range pesticide resistance, the spread of H. armigera in the New World may have been hastened by multiple introductions into South America and/or the Caribbean. The recent expansion of the range of H. armigera into the New World is analyzed herein using mtDNA of samples from South America, the Caribbean Basin, and the Florida Peninsula. Phylogeographic analyses reveal that several haplotypes are nearly ubiquitous throughout the New World and native range of H. armigera, but several haplotypes have limited geographic distribution from which a secondary introduction with Euro-African origins into the New World is inferred. In addition, host–haplotype correlations were analyzed to see whether haplotypes might be restricted to certain crops. No specialization was found; however, some haplotypes had a broader host range than others. These results suggest that the dispersal of H. armigera in the New World is occurring from both natural migration and human-mediated introductions. As such, both means of introduction should be monitored to prevent the spread of H. armigera into areas such as the United States, Mexico, and Canada, where it is not yet established.

The beta-site APP cleaving enzyme 1, known as BACE1, has been a widely pursued Alzheimer's disease drug target owing to its critical role in the production of amyloid-beta. We have previously reported the clinical development of LY2811376 and LY2886721. LY2811376 advanced to Phase I before development was terminated due to nonclinical retinal toxicity. LY2886721 advanced to Phase II, but development was halted due to abnormally elevated liver enzymes. Herein, we report the discovery and clinical development of LY3202626, a highly potent, CNS-penetrant, and low-dose BACE inhibitor, which successfully addressed these key development challenges.

The application of DNA barcoding represents a complementary and efficient approach to identifying specimens at all stages of their life cycle when used in combination with traditional morphological methods. Due to difficulties obtaining samples from the deep sea (> 200 m), these methods have been less frequently applied to deep-water taxa. We used DNA-barcoding techniques to enhance large-scale biodiversity initiatives for deep-pelagic crustaceans within the Gulf of Mexico, a region that has recently been identified as one of the world’s four most hyperdiverse ocean ecosystems. This study was conceptualized in direct response to the Deepwater Horizon Oil Spill in 2010, which identified major knowledge gaps in our understanding of deep-sea biodiversity. We employed traditional Sanger sequencing and a genomic skimming approach to target the mitochondrial ribosomal large subunit 16S and the protein-coding cytochrome oxidase subunit 1 (COI). Alongside these molecular approaches, traditional taxonomic investigations allowed for advancements in biodiversity, evolutionary relationships, cryptic species complexes, and distributional records across four abundant and common deep-pelagic orders (Amphipoda, Euphausiacea, Lophogastrida, and Decapoda). DNA barcodes were successfully obtained from 82 species for a total of 158 and 169 new 16S and COI sequences, respectively. Evidence of cryptic diversity has been found in the genera EucopiaDana, 1852 (Lophogastrida) and Allosergestes Judkins & Kensley, 2008 (Decapoda). New records for the Gulf of Mexico of species of LanceolaSay, 1818 (Amphipoda), Eupasiphae Wood-Mason in Wood-Mason & Alcock, 1893, PasiphaeaSavigny, 1816, and MeningodoraSmith, 1882 (Caridea) are presented. Preliminary results allow us to reconsider the current classification and evolutionary relationships of several lineages. The urgency to document biodiversity in the deep-pelagic is pressing against a backdrop of future threats including oil spills and deep-sea drilling.

PRMT5 and its substrate adaptor proteins (SAPs), pICln and Riok1, are synthetic lethal dependencies in MTAP-deleted cancer cells. SAPs share a conserved PRMT5 binding motif (PBM) which mediates binding to a surface of PRMT5 distal to the catalytic site. This interaction is required for methylation of several PRMT5 substrates, including histone and spliceosome complexes. We screened for small molecule inhibitors of the PRMT5-PBM interaction and validated a compound series which binds to the PRMT5-PBM interface and directly inhibits binding of SAPs. Mode of action and structure determination studies revealed that these compounds form a covalent bond between a halogenated pyridazinone group and cysteine 278 of PRMT5. Optimization of the starting hit produced a lead compound, BRD0639, which engages the target in cells, disrupts the PRMT5-RIOK1 complex, and reduces substrate methylation. BRD0639 is a first-in-class PBM-competitive small molecule that can support studies of PBM-dependent PRMT5 activities and the development of novel PRMT5 inhibitors that selectively target these functions.

Genomic approaches hold great promise for resolving unanswered questions about transmission patterns and responses to control efforts for schistosomiasis and other neglected tropical diseases. However, the cost of generating genomic data and the challenges associated with obtaining sufficient DNA from individual schistosome larvae (miracidia) from mammalian hosts have limited the application of genomic data for studying schistosomes and other complex macroparasites. Here, we demonstrate the feasibility of utilizing whole genome amplification and sequencing (WGS) to analyze individual archival miracidia. As an example, we sequenced whole genomes of 22 miracidia from 11 human hosts representing two villages in rural Sichuan, China, and used these data to evaluate patterns of relatedness and genetic diversity. We also down-sampled our dataset to test how lower coverage sequencing could increase the cost effectiveness of WGS while maintaining power to accurately infer relatedness. Collectively, our results illustrate that population-level WGS datasets are attainable for individual miracidia and represent a powerful tool for ultimately providing insight into overall genetic diversity, parasite relatedness, and transmission patterns for better design and evaluation of disease control efforts., Author summary Schistosomiasis is a neglected tropical disease caused by helminths within the genus Schistosoma, which affects over 200 million people globally. Here, we demonstrate the ability to obtain whole genome sequences from single S. japonicum miracidia that have been archived for years, and the promise of how these data may be used to answer major questions to understand and eventually help control schistosomiasis. To demonstrate the approach, we generated a moderate-sized dataset of 22 individual S. japonicum genomes and use these data to infer fine-scale patterns of relatedness among parasites from human hosts from two villages in Sichuan, China. We highlight how this type of genomic information may be useful for understanding the genetic variation among individual parasites with high precision, which could ultimately provide high resolution for understanding transmission pathways and inform control efforts with larger sample sizes. We also show that it is possible to use less data per individual than we obtained here, and still maintain high levels of accuracy; this would enable greater sample sizes by lowering individual sample cost. Collectively our results demonstrate that sampling genomes of individual schistosome parasites is now feasible on a population-level scale, opening new avenues for studying relatedness, tracing parasite spread, and studying parasite biology.

From its inception, population genetics has been nearly as concerned with the genetic data type—to which analyses are brought to bear—as it is with the analysis methods themselves. The field has traversed allozymes, microsatellites, segregating sites in multilocus alignments and, currently, single nucleotide polymorphisms (SNPs) generated by high‐throughput genomic sequencing methods, primarily whole genome sequencing and reduced representation library (RRL) sequencing. As each emerging data type has gained traction, it has been compared to existing methods, based on its relative ability to discern population structural complexity at increasing levels of resolution. However, this is usually done by comparing the gold standard in one data type to the gold standard in the new data type. These gold standards frequently differ in power and in sampling density, both across a genome and throughout a spatial range. In this issue of Molecular Ecology, D’Aloia et al. apply the high‐throughput approach as fully as possible to microsatellites, nuclear loci and SNPs genotyped through an RRL method; this is coupled with a spatially dense sampling scheme. Completing a battery of population genetics analyses across data types (including a series of down‐sampled data sets), the authors find that SNP data are slightly more sensitive to fine‐scale genetic structure, and the results are more resilient to down‐sampling than microsatellites and nonrepetitive nuclear loci. However, their results are far from an unqualified victory for RRL SNP data over all previous data types: the authors note that modest additions to the microsatellites and nuclear loci data sets may provide the necessary analytical power to delineate the fine‐scale genetic structuring identified by SNPs. As always, as the field begins to fully embrace the newest thing, good science reminds us that traditional data types are far from useless, especially when combined with a well‐designed sampling scheme.

The Gulf of Mexico experiences frequent perturbations, both natural and anthropogenic. To better understand the impacts of these events, we must inventory natural variability within the ecosystem, communities, species, and populations, and contextualize these findings in relation to physical features. Here, we present an integrated study of comparative population genomics and biophysical oceanography. Targeting three species of mesopelagic shrimp common to the Gulf of Mexico midwater (Acanthephyra purpurea, Systellaspis debilis, and Robustosergia robusta), we analyzed genetic diversity and population connectivity as proxies for species health and resilience, respectively. We also simulated a range of vertical migratory behaviors for the shrimp to infer the relationship between diel vertical migration and horizontal transmission between the Gulf of Mexico and the greater Atlantic Ocean. This study aims to establish biological baselines and characterize these values in terms of the prevailing oceanographic feature of the midwater: the Gulf Loop Current. Generally, the oplophorid species (A. purpurea and S. debilis) exhibit lower genetic diversity and higher interpopulation homogeneity compared to the sergestid (R. robusta). Biophysical simulations suggest the differences in vertical migratory regimes between these two groups have important implications for horizontal transport out of the Gulf of Mexico. Because of the difference in vertical migration patterns, access to the Gulf Loop Current varies across taxa and impacts inter-basin migration. Our findings suggest a negative correlation between surface abundance and genetic diversity in these three shrimp species. We hypothesize that this correlation may be due to the relationships between surface abundance and access to the fastest moving waters of the Gulf Loop Current.

Cryogenic electron microscopy (cryo-EM) has become an enabling technology in drug discovery and in understanding molecular bases of disease by producing near-atomic resolution (less than 0.4 nm) 3D reconstructions of biological macro-molecules. The imaging process required for 3D reconstructions involves a highly iterative and empirical screening process, starting with the acquisition of low magnification images of the cryo-EM grids. These images are inspected for squares that are likely to contain useful molecular signals. Potentially useful squares within the grid are then imaged at progressively higher magnifications, with the goal of identifying sub-micron areas within circular holes (bounded by the squares) for imaging at high magnification. This arduous, multi-step data acquisition process represents a bottleneck for obtaining a high throughput data collection. Here, we focus on automating the early decision making for the microscope operator, scoring low magnification images of squares, and proposing the first deep learning framework, XCryoNet, for automated cryo-EM grid screening. XCryoNet is a semi-supervised, attention-guided deep learning approach that provides explainable scoring of automatically extracted square images using limited amounts of labeled data. Results show up to 8% and 37% improvements over a fully supervised and a no-attention solution, respectively, when labeled data is scarce.

The deep waters of the open ocean represent a major frontier in exploration and scientific understanding. However, modern technological and computational tools are making the deep ocean more accessible than ever before by facilitating increasingly sophisticated studies of deep ocean ecosystems. Here, we describe some of the cutting-edge technologies that have been employed by the Deep Pelagic Nekton Dynamics of the Gulf of Mexico (DEEPEND; www.deependconsortium.org) Consortium to study the biodiverse fauna and dynamic physical-chemical environment of the offshore Gulf of Mexico (GoM) from 0 to 1,500 m.

Population genetics has gained popularity as a method to discover glacial refugia in terrestrial species, but has only recently been applied to the marine realm. The last glacial maxima occurred 20,000 years ago, decreasing sea levels by 120 m and exposing much of the continental shelf in the northern Gulf of Mexico, with the exception of De Soto Canyon (2100 m depth). The goal of this study was to determine whether population dynamics of the giant deep-sea isopod, Bathynomus giganteus, were better explained by habitat diversity or by the past presence of a marine glacial refugium in De Soto Canyon. To accomplish this we (1) measured genetic diversity in De Soto Canyon and adjacent regions, (2) characterized gene flow and connectivity between these regions, and (3) investigated historical changes to population size. We sequenced three mitochondrial loci (12S, 16S, and COI) from 212 individuals and also performed a next-generation sequencing pilot study using double digest Restriction site-Associated DNA sequencing. We found high genetic diversity and connectivity throughout the study regions, migration between all three regions, low population differentiation, and evidence of population expansion. This study suggests that habitat heterogeneity, rather than the presence of a glacial refugium, has had an historical effect on the population dynamics of B. giganteus.

The silver Y moth [Autographa gamma (Linneaus) (Noctuidae: Plusiinae)] is a pervasive crop pest in its native range but has not been found in moth surveys in the United States. Specimens of A. gamma are often intercepted at U.S. ports of entry, so the risk of introduction of this invasive species is high. Currently, identification of Plusiinae adults captured in domestic surveys is done by morphlogical comparison; however, this method is time consuming and misidentifications have occurred in the past. A recent study outlined a real-time PCR assay capable of rapidly identifying individual A. gamma specimens using CO1. This same study provided preliminary data for a droplet digital PCR (ddPCR) assay capable of processing bulk trap samples. Here, we develop and test a ddPCR assay for detecting a single A. gamma in a trap sample of 200 individual moths. This assay will drastically reduce the time and cost needed to screen domestic trap samples for A. gamma.

Merestinib is an oral multi-kinase inhibitor targeting a limited number of oncokinases including MET, AXL, RON and MKNK1/2. Here, we report that merestinib inhibits neurotrophic receptor tyrosine kinases NTRK1/2/3 which are oncogenic drivers in tumors bearing NTRK fusion resulting from chromosomal rearrangements. Merestinib is shown to be a type II NTRK1 kinase inhibitor as determined by x-ray crystallography. In KM-12 cells harboring TPM3-NTRK1 fusion, merestinib exhibits potent p-NTRK1 inhibition in vitro by western blot and elicits an anti-proliferative response in two- and three-dimensional growth. Merestinib treatment demonstrated profound tumor growth inhibition in in vivo cancer models harboring either a TPM3-NTRK1 or an ETV6-NTRK3 gene fusion. To recapitulate resistance observed from type I NTRK kinase inhibitors entrectinib and larotrectinib, we generated NIH-3T3 cells exogenously expressing TPM3-NTRK1 wild-type, or acquired mutations G595R and G667C in vitro and in vivo. Merestinib blocks tumor growth of both wild-type and mutant G667C TPM3-NTRK1 expressing NIH-3T3 cell-derived tumors. These preclinical data support the clinical evaluation of merestinib, a type II NTRK kinase inhibitor (NCT02920996), both in treatment naïve patients and in patients progressed on type I NTRK kinase inhibitors with acquired secondary G667C mutation in NTRK fusion bearing tumors.