LXR/ApoE Activation Restricts Innate Immune Suppression in Cancer.

Therapeutic harnessing of adaptive immunity via checkpoint inhibition has transformed the treatment of many cancers. Despite unprecedented long-term responses, most patients do not respond to these therapies. Immunotherapy non-responders often harbor high levels of circulating myeloid-derived suppressor cells (MDSCs)-an immunosuppressive innate cell population. Through genetic and pharmacological approaches, we uncovered a pathway governing MDSC abundance in multiple cancer types. Therapeutic liver-X nuclear receptor (LXR) agonism reduced MDSC abundance in murine models and in patients treated in a first-in-human dose escalation phase 1 trial. MDSC depletion was associated with activation of cytotoxic T lymphocyte (CTL) responses in mice and patients. The LXR transcriptional target ApoE mediated these effects in mice, where LXR/ApoE activation therapy elicited robust anti-tumor responses and also enhanced T cell activation during various immune-based therapies. We implicate the LXR/ApoE axis in the regulation of innate immune suppression and as a target for enhancing the efficacy of cancer immunotherapy in patients.

Chemistry-First Approach for Nomination of Personalized Treatment in Lung Cancer.

Diversity in the genetic lesions that cause cancer is extreme. In consequence, a pressing challenge is the development of drugs that target patient-specific disease mechanisms. To address this challenge, we employed a chemistry-first discovery paradigm for de novo identification of druggable targets linked to robust patient selection hypotheses. In particular, a 200,000 compound diversity-oriented chemical library was profiled across a heavily annotated test-bed of >100 cellular models representative of the diverse and characteristic somatic lesions for lung cancer. This approach led to the delineation of 171 chemical-genetic associations, shedding light on the targetability of mechanistic vulnerabilities corresponding to a range of oncogenotypes present in patient populations lacking effective therapy. Chemically addressable addictions to ciliogenesis in TTC21B mutants and GLUT8-dependent serine biosynthesis in KRAS/KEAP1 double mutants are prominent examples. These observations indicate a wealth of actionable opportunities within the complex molecular etiology of cancer.

Metabolic Heterogeneity in Human Lung Tumors.

Non-small cell lung cancer (NSCLC) is heterogeneous in the genetic and environmental parameters that influence cell metabolism in culture. Here, we assessed the impact of these factors on human NSCLC metabolism in vivo using intraoperative (13)C-glucose infusions in nine NSCLC patients to compare metabolism between tumors and benign lung. While enhanced glycolysis and glucose oxidation were common among these tumors, we observed evidence for oxidation of multiple nutrients in each of them, including lactate as a potential carbon source. Moreover, metabolically heterogeneous regions were identified within and between tumors, and surprisingly, our data suggested potential contributions of non-glucose nutrients in well-perfused tumor areas. Our findings not only demonstrate the heterogeneity in tumor metabolism in vivo but also highlight the strong influence of the microenvironment on this feature.

Systematic identification of molecular subtype-selective vulnerabilities in non-small-cell lung cancer.

Context-specific molecular vulnerabilities that arise during tumor evolution represent an attractive intervention target class. However, the frequency and diversity of somatic lesions detected among lung tumors can confound efforts to identify these targets. To confront this challenge, we have applied parallel screening of chemical and genetic perturbations within a panel of molecularly annotated NSCLC lines to identify intervention opportunities tightly linked to molecular response indicators predictive of target sensitivity. Anchoring this analysis on a matched tumor/normal cell model from a lung adenocarcinoma patient identified three distinct target/response-indicator pairings that are represented with significant frequencies (6%-16%) in the patient population. These include NLRP3 mutation/inflammasome activation-dependent FLIP addiction, co-occurring KRAS and LKB1 mutation-driven COPI addiction, and selective sensitivity to a synthetic indolotriazine that is specified by a seven-gene expression signature. Target efficacies were validated in vivo, and mechanism-of-action studies informed generalizable principles underpinning cancer cell biology.

Metabolic Diversity in Human Non-Small Cell Lung Cancer Cells.

Intermediary metabolism in cancer cells is regulated by diverse cell-autonomous processes, including signal transduction and gene expression patterns, arising from specific oncogenotypes and cell lineages. Although it is well established that metabolic reprogramming is a hallmark of cancer, we lack a full view of the diversity of metabolic programs in cancer cells and an unbiased assessment of the associations between metabolic pathway preferences and other cell-autonomous processes. Here, we quantified metabolic features, mostly from the 13C enrichment of molecules from central carbon metabolism, in over 80 non-small cell lung cancer (NSCLC) cell lines cultured under identical conditions. Because these cell lines were extensively annotated for oncogenotype, gene expression, protein expression, and therapeutic sensitivity, the resulting database enables the user to uncover new relationships between metabolism and these orthogonal processes.

A stress-induced tyrosine-tRNA depletion response mediates codon-based translational repression and growth suppression.

Eukaryotic transfer RNAs can become selectively fragmented upon various stresses, generating tRNA-derived small RNA fragments. Such fragmentation has been reported to impact a small fraction of the tRNA pool and thus presumed to not directly impact translation. We report that oxidative stress can rapidly generate tyrosine-tRNAGUA fragments in human cells-causing significant depletion of the precursor tRNA. Tyrosine-tRNAGUA depletion impaired translation of growth and metabolic genes enriched in cognate tyrosine codons. Depletion of tyrosine tRNAGUA or its translationally regulated targets USP3 and SCD repressed proliferation-revealing a dedicated tRNA-regulated growth-suppressive pathway for oxidative stress response. Tyrosine fragments are generated in a DIS3L2 exoribonuclease-dependent manner and inhibit hnRNPA1-mediated transcript destabilization. Moreover, tyrosine fragmentation is conserved in C. elegans. Thus, tRNA fragmentation can coordinately generate trans-acting small RNAs and functionally deplete a tRNA. Our findings reveal the existence of an underlying adaptive codon-based regulatory response inherent to the genetic code.

High-throughput functional annotation of natural products by integrated activity profiling.

Determining mechanism of action (MOA) is one of the biggest challenges in natural products discovery. Here, we report a comprehensive platform that uses Similarity Network Fusion (SNF) to improve MOA predictions by integrating data from the cytological profiling high-content imaging platform and the gene expression platform Functional Signature Ontology, and pairs these data with untargeted metabolomics analysis for de novo bioactive compound discovery. The predictive value of the integrative approach was assessed using a library of target-annotated small molecules as benchmarks. Using Kolmogorov-Smirnov (KS) tests to compare in-class to out-of-class similarity, we found that SNF retains the ability to identify significant in-class similarity across a diverse set of target classes, and could find target classes not detectable in either platform alone. This confirmed that integration of expression-based and image-based phenotypes can accurately report on MOA. Furthermore, we integrated untargeted metabolomics of complex natural product fractions with the SNF network to map biological signatures to specific metabolites. Three examples are presented where SNF coupled with metabolomics was used to directly functionally characterize natural products and accelerate identification of bioactive metabolites, including the discovery of the azoxy-containing biaryl compounds parkamycins A and B. Our results support SNF integration of multiple phenotypic screening approaches along with untargeted metabolomics as a powerful approach for advancing natural products drug discovery.

Modulation of prey capture kinematics in relation to prey distance helps predict success.

Predators are not perfect, as some of their prey capture attempts result in failure. Successful attempts may be partly due to predators modulating their capture kinematics in relation to variation in the visual cues of the prey to increase the probability of success. In praying mantises, which have been suggested to possess stereoscopic vision, variation in prey distance has been shown to elicit variation in the probability of an attempt. However, it remains to be examined whether variation in prey distance results in mantises modulating their attempt to successfully capture prey. The goals of this study were to examine these relationships using the praying mantis system. Using 11 adult female Sphodromantis lineola, we recorded 192 prey capture attempts at 1000 Hz with two cameras to examine the 3D kinematics of successful and unsuccessful prey capture attempts. Using a combination of principal component analysis (PCA) and logistic regression, our results show that as prey distance increases, mantises adjust through greater and faster expansion of the forelegs and body (PC1), which significantly predicts capture success. However, PC1 only explains 22% of the variation in all prey capture attempts, suggesting that the other components may be related to additional aspects of the prey. Our results suggest that the distances at which mantises prefer to attempt to capture prey may be the result of their greater probability of successfully capturing the prey. These results highlight the range of motions mantises use when attempting to capture prey, suggesting flexibility in their prey capture attempts in relation to prey position.

KAP1 Recruitment of the 7SK snRNP Complex to Promoters Enables Transcription Elongation by RNA Polymerase II.

The transition from transcription initiation to elongation at promoters of primary response genes (PRGs) in metazoan cells is controlled by inducible transcription factors, which utilize P-TEFb to phosphorylate RNA polymerase II (Pol II) in response to stimuli. Prior to stimulation, a fraction of P-TEFb is recruited to promoter-proximal regions in a catalytically inactive state bound to the 7SK small nuclear ribonucleoprotein (snRNP) complex. However, it remains unclear how and why the 7SK snRNP is assembled at these sites. Here we report that the transcriptional regulator KAP1 continuously tethers the 7SK snRNP to PRG promoters to facilitate P-TEFb recruitment and productive elongation in response to stimulation. Remarkably, besides PRGs, genome-wide studies revealed that KAP1 and 7SK snRNP co-occupy most promoter-proximal regions containing paused Pol II. Collectively, we provide evidence of an unprecedented mechanism controlling 7SK snRNP delivery to promoter-proximal regions to facilitate "on-site" P-TEFb activation and Pol II elongation.

Polymerase chain reaction for the in vitro detection of the pESI plasmid associated with the globally circulating Salmonella Infantis outbreak strain.

A globally circulating strain of Salmonella enterica serotype Infantis containing the pESI plasmid has increased in prevalence in poultry meat samples and cases of human infections. In this study, a polymerase chain reaction (PCR) protocol was designed to detect the pESI plasmid and confirm the Infantis serotype of Salmonella isolates. Primers were tested bioinformatically to predict specificity, sensitivity, and precision. A total of 54 isolates of Salmonella serotypes Infantis, Senftenberg, and Alachua were tested, with and without the pESI plasmid carriage. Isolates of 31 additional serotypes were also screened to confirm specificity to Infantis. Specificity, sensitivity, and precision of each primer were >0.95. All isolates tested produced the expected band sizes. This PCR protocol provides a rapid and clear result for the detection of the pESI plasmid and serotype Infantis and will allow for the in vitro detection for epidemiological studies where whole-genome sequencing is not available.

Analysis of Salmonella enterica Isolated from a Mixed-Use Watershed in Georgia, USA: Antimicrobial Resistance, Serotype Diversity, and Genetic Relatedness to Human Isolates.

As the cases of Salmonella enterica infections associated with contaminated water are increasing, this study was conducted to address the role of surface water as a reservoir of S. enterica serotypes. We sampled rivers and streams (n = 688) over a 3-year period (2015 to 2017) in a mixed-use watershed in Georgia, USA, and 70.2% of the total stream samples tested positive for Salmonella. A total of 1,190 isolates were recovered and characterized by serotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis (PFGE). A wide range of serotypes was identified, including those commonly associated with humans and animals, with S. enterica serotype Muenchen being predominant (22.7%) and each serotype exhibiting a high degree of strain diversity by PFGE. About half (46.1%) of the isolates had PFGE patterns indistinguishable from those of human clinical isolates in the CDC PulseNet database. A total of 52 isolates (4.4%) were resistant to antimicrobials, out of which 43 isolates were multidrug resistant (MDR; resistance to two or more classes of antimicrobials). These 52 resistant Salmonella isolates were screened for the presence of antimicrobial resistance genes, plasmid replicons, and class 1 integrons, out of which four representative MDR isolates were selected for whole-genome sequencing analysis. The results showed that 28 MDR isolates resistant to 10 antimicrobials had blacmy-2 on an A/C plasmid. Persistent contamination of surface water with a high diversity of Salmonella strains, some of which are drug resistant and genetically indistinguishable from human isolates, supports a role of environmental surface water as a reservoir for and transmission route of this pathogen. IMPORTANCE Salmonella has been traditionally considered a foodborne pathogen, as it is one of the most common etiologies of foodborne illnesses worldwide; however, recent Salmonella outbreaks attributed to fresh produce and water suggest a potential environmental source of Salmonella that causes some human illnesses. Here, we investigated the prevalence, diversity, and antimicrobial resistance of Salmonella isolated from a mixed-use watershed in Georgia, USA, in order to enhance the overall understanding of waterborne Salmonella. The persistence and widespread distribution of Salmonella in surface water confirm environmental sources of the pathogen. A high proportion of waterborne Salmonella with clinically significant serotypes and genetic similarity to strains of human origin supports the role of environmental water as a significant reservoir of Salmonella and indicates a potential waterborne transmission of Salmonella to humans. The presence of antimicrobial-resistant and MDR Salmonella demonstrates additional risks associated with exposure to contaminated environmental water.

XPO1-dependent nuclear export is a druggable vulnerability in KRAS-mutant lung cancer.

The common participation of oncogenic KRAS proteins in many of the most lethal human cancers, together with the ease of detecting somatic KRAS mutant alleles in patient samples, has spurred persistent and intensive efforts to develop drugs that inhibit KRAS activity. However, advances have been hindered by the pervasive inter- and intra-lineage diversity in the targetable mechanisms that underlie KRAS-driven cancers, limited pharmacological accessibility of many candidate synthetic-lethal interactions and the swift emergence of unanticipated resistance mechanisms to otherwise effective targeted therapies. Here we demonstrate the acute and specific cell-autonomous addiction of KRAS-mutant non-small-cell lung cancer cells to receptor-dependent nuclear export. A multi-genomic, data-driven approach, utilizing 106 human non-small-cell lung cancer cell lines, was used to interrogate 4,725 biological processes with 39,760 short interfering RNA pools for those selectively required for the survival of KRAS-mutant cells that harbour a broad spectrum of phenotypic variation. Nuclear transport machinery was the sole process-level discriminator of statistical significance. Chemical perturbation of the nuclear export receptor XPO1 (also known as CRM1), with a clinically available drug, revealed a robust synthetic-lethal interaction with native or engineered oncogenic KRAS both in vitro and in vivo. The primary mechanism underpinning XPO1 inhibitor sensitivity was intolerance to the accumulation of nuclear IκBα (also known as NFKBIA), with consequent inhibition of NFκB transcription factor activity. Intrinsic resistance associated with concurrent FSTL5 mutations was detected and determined to be a consequence of YAP1 activation via a previously unappreciated FSTL5-Hippo pathway regulatory axis. This occurs in approximately 17% of KRAS-mutant lung cancers, and can be overcome with the co-administration of a YAP1-TEAD inhibitor. These findings indicate that clinically available XPO1 inhibitors are a promising therapeutic strategy for a considerable cohort of patients with lung cancer when coupled to genomics-guided patient selection and observation.

Serotyping of sub-Saharan Africa Salmonella strains isolated from poultry feces using multiplex PCR and whole genome sequencing.

BACKGROUND: Salmonella enterica remains a leading cause of food-borne diseases worldwide. Serotype information is important in food safety and public health activities to reduce the burden of salmonellosis. In the current study, two methods were used to determine serotypes of 111 strains of Salmonella isolated from poultry feces in Burkina Faso. First, Salmonella Multiplex Assay for Rapid Typing (SMART) Polymerase Chain Reaction (PCR) was used to determine the serovars of the S. enterica isolates. Second, serovar prediction based on whole genome sequencing (WGS) data was performed using SeqSero 2.0. RESULTS: Among the 111 Salmonella isolates, serotypes for 17 (15.31%) isolates were identified based on comparison to a panel of representative SMART codes previously determined for the 50 most common serovars in the United States. Forty-four (44) new SMART codes were developed for common and uncommon serotypes. A total of 105 (94.59%) isolates were serotyped using SeqSero 2.0 for serovar prediction based on WGS data. CONCLUSION: We determined that SeqSero 2.0 was more comprehensive for identifying Salmonella serotypes from Burkina Faso than SMART PCR.

To Scan or Not to Scan: Development of a Clinical Decision Support Tool to Determine if Imaging Would Aid in the Diagnosis of Appendicitis.

BACKGROUND: Appendicitis is one of the most common surgically treated diseases in the world. CT scans are often over-utilized and ordered before a surgeon has evaluated the patient. Our aim was to develop a tool using machine learning (ML) algorithms that would help determine if there would be benefit in obtaining a CT scan prior to surgeon consultation. METHODS: Retrospective chart review of 100 randomly selected cases who underwent appendectomy and 100 randomly selected controls was completed. Variables included components of the patient's history, laboratory values, CT readings, and pathology. Pathology was used as the gold standard for appendicitis diagnosis. All variables were then used to build the ML algorithms. Random Forest (RF), Support Vector Machine (SVM), and Bayesian Network Classifiers (BNC) models with and without CT scan results were trained and compared to CT scan results alone and the Alvarado score using area under the Receiver Operator Curve (ROC), sensitivity, and specificity measures as well as calibration indices from 500 bootstrapped samples. RESULTS: Among the cases that underwent appendectomy, 88% had pathology-confirmed appendicitis. All the ML algorithms had better sensitivity, specificity, and ROC than the Alvarado score. SVM with and without CT had the best indices and could predict if imaging would aid in appendicitis diagnosis. CONCLUSION: This study demonstrated that SVM with and without CT results can be used for selective imaging in the diagnosis of appendicitis. This study serves as the initial step and proof-of-concept to externally validate these results with larger and more diverse patient population.

Computational discovery of pathway-level genetic vulnerabilities in non-small-cell lung cancer.

MOTIVATION: Novel approaches are needed for discovery of targeted therapies for non-small-cell lung cancer (NSCLC) that are specific to certain patients. Whole genome RNAi screening of lung cancer cell lines provides an ideal source for determining candidate drug targets. RESULTS: Unsupervised learning algorithms uncovered patterns of differential vulnerability across lung cancer cell lines to loss of functionally related genes. Such genetic vulnerabilities represent candidate targets for therapy and are found to be involved in splicing, translation and protein folding. In particular, many NSCLC cell lines were especially sensitive to the loss of components of the LSm2-8 protein complex or the CCT/TRiC chaperonin. Different vulnerabilities were also found for different cell line subgroups. Furthermore, the predicted vulnerability of a single adenocarcinoma cell line to loss of the Wnt pathway was experimentally validated with screening of small-molecule Wnt inhibitors against an extensive cell line panel. AVAILABILITY AND IMPLEMENTATION: The clustering algorithm is implemented in Python and is freely available at https://bitbucket.org/youngjh/nsclc_paper CONTACT: marcotte@icmb.utexas.edu or jon.young@utexas.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Mode of action and pharmacogenomic biomarkers for exceptional responders to didemnin B.

Modern cancer treatment employs many effective chemotherapeutic agents originally discovered from natural sources. The cyclic depsipeptide didemnin B has demonstrated impressive anticancer activity in preclinical models. Clinical use has been approved but is limited by sparse patient responses combined with toxicity risk and an unclear mechanism of action. From a broad-scale effort to match antineoplastic natural products to their cellular activities, we found that didemnin B selectively induces rapid and wholesale apoptosis through dual inhibition of PPT1 and EEF1A1. Furthermore, empirical discovery of a small panel of exceptional responders to didemnin B allowed the generation of a regularized regression model to extract a sparse-feature genetic biomarker capable of predicting sensitivity to didemnin B. This may facilitate patient selection in a fashion that could enhance and expand the therapeutic application of didemnin B against neoplastic disease.

A genome-scale screen reveals context-dependent ovarian cancer sensitivity to miRNA overexpression.

Large-scale molecular annotation of epithelial ovarian cancer (EOC) indicates remarkable heterogeneity in the etiology of that disease. This diversity presents a significant obstacle against intervention target discovery. However, inactivation of miRNA biogenesis is commonly associated with advanced disease. Thus, restoration of miRNA activity may represent a common vulnerability among diverse EOC oncogenotypes. To test this, we employed genome-scale, gain-of-function, miRNA mimic toxicity screens in a large, diverse spectrum of EOC cell lines. We found that all cell lines responded to at least some miRNA mimics, but that the nature of the miRNA mimics provoking a response was highly selective within the panel. These selective toxicity profiles were leveraged to define modes of action and molecular response indicators for miRNA mimics with tumor-suppressive characteristics in vivo. A mechanistic principle emerging from this analysis was sensitivity of EOC to miRNA-mediated release of cell fate specification programs, loss of which may be a prerequisite for development of this disease.

Genome analysis of Salmonella enterica serovar enteritis strains isolated from poultry and humans in Burkina Faso.

Whole-genome sequencing (WGS) was used to characterize four Salmonella enterica Enteritidis isolates from poultry (n=2) and human (n=2) from Ouagadougou, Burkina Faso. Antimicrobial resistance genes, chromosomal mutations, and mobile genetic elements were identified by analysis of WGS data using sequence homology.

Exudate From Retail Chicken Liver Packaging Allows for Survival of Naturally Occurring Campylobacter, Coliforms, and Aerobic Microorganisms Under Drying Conditions.

Campylobacter spp. are a leading cause of human foodborne illness associated with chicken meat products in the United States. Chicken livers, including exudate from packaging, commonly carry Campylobacter and could be a source of illness if mishandled. Survivability of naturally occurring Campylobacter, total aerobic bacteria, and coliforms was determined under drying conditions in two consumer simulated environments: moist sponge and solid surface. Fresh chicken liver exudate was dispensed onto sponges and glass slides and allowed to dry under ambient conditions for 7 days. Bacterial concentration was measured at 0, 6, 24, 48, 72, and 168 h. Total aerobic population did not decrease by more than one log over 7 days and did not correlate to water activity or time in either simulation. Coliform concentrations increased in sponge simulations but decreased in solid surface simulations. Further, coliform concentrations were significantly higher in sponge simulations than in solid surface. Campylobacter was naturally present in exudate and survived at least to 6 h in every trial. Campylobacter was recoverable at 24 h in some sponge trials. However, Campylobacter concentration was strongly correlated to water activity. Fresh chicken liver exudate could present a risk of campylobacteriosis to consumers if mishandled even after drying.