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.

Degradation of AMPK by a cancer-specific ubiquitin ligase.

AMP-activated protein kinase (AMPK) is a master sensor and regulator of cellular energy status. Upon metabolic stress, AMPK suppresses anabolic and promotes catabolic processes to regain energy homeostasis. Cancer cells can occasionally suppress the growth-restrictive AMPK pathway by mutation of an upstream regulatory kinase. Here, we describe a widespread mechanism to suppress AMPK through its ubiquitination and degradation by the cancer-specific MAGE-A3/6-TRIM28 ubiquitin ligase. MAGE-A3 and MAGE-A6 are highly similar proteins normally expressed only in the male germline but frequently re-activated in human cancers. MAGE-A3/6 are necessary for cancer cell viability and are sufficient to drive tumorigenic properties of non-cancerous cells. Screening for targets of MAGE-A3/6-TRIM28 revealed that it ubiquitinates and degrades AMPKα1. This leads to inhibition of autophagy, activation of mTOR signaling, and hypersensitization to AMPK agonists, such as metformin. These findings elucidate a germline mechanism commonly hijacked in cancer to suppress AMPK.

Identification of direct connections between the dura and the brain.

The arachnoid barrier delineates the border between the central nervous system and dura mater. Although the arachnoid barrier creates a partition, communication between the central nervous system and the dura mater is crucial for waste clearance and immune surveillance1,2. How the arachnoid barrier balances separation and communication is poorly understood. Here, using transcriptomic data, we developed transgenic mice to examine specific anatomical structures that function as routes across the arachnoid barrier. Bridging veins create discontinuities where they cross the arachnoid barrier, forming structures that we termed arachnoid cuff exit (ACE) points. The openings that ACE points create allow the exchange of fluids and molecules between the subarachnoid space and the dura, enabling the drainage of cerebrospinal fluid and limited entry of molecules from the dura to the subarachnoid space. In healthy human volunteers, magnetic resonance imaging tracers transit along bridging veins in a similar manner to access the subarachnoid space. Notably, in neuroinflammatory conditions such as experimental autoimmune encephalomyelitis, ACE points also enable cellular trafficking, representing a route for immune cells to directly enter the subarachnoid space from the dura mater. Collectively, our results indicate that ACE points are a critical part of the anatomy of neuroimmune communication in both mice and humans that link the central nervous system with the dura and its immunological diversity and waste clearance systems.

GWAS meets TCGA to illuminate mechanisms of cancer predisposition.

Genome-wide association studies (GWASs) have unraveled a large number of cancer risk alleles. Understanding how these allelic variants predispose to disease is a major bottleneck confronting translational application. In this issue, Li and colleagues combine GWASs with The Cancer Genome Atlas (TCGA) to disambiguate the contributions of germline and somatic variants to tumorigenic gene expression programs. They find that close to half of the known risk alleles for estrogen receptor (ER)-positive breast cancer are expression quantitative trait loci (eQTLs) acting upon major determinants of gene expression in tumors.

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.

Pathogenic variants in CRX have distinct cis-regulatory effects on enhancers and silencers in photoreceptors.

Dozens of variants in the gene for the homeodomain transcription factor (TF) cone-rod homeobox (CRX) are linked with human blinding diseases that vary in their severity and age of onset. How different variants in this single TF alter its function in ways that lead to a range of phenotypes is unclear. We characterized the effects of human disease-causing variants on CRX cis-regulatory function by deploying massively parallel reporter assays (MPRAs) in mouse retina explants carrying knock-ins of two variants, one in the DNA-binding domain (p.R90W) and the other in the transcriptional effector domain (p.E168d2). The degree of reporter gene dysregulation in these mutant Crx retinas corresponds with their phenotypic severity. The two variants affect similar sets of enhancers, and p.E168d2 has distinct effects on silencers. Cis-regulatory elements (CREs) near cone photoreceptor genes are enriched for silencers that are derepressed in the presence of p.E168d2. Chromatin environments of CRX-bound loci are partially predictive of episomal MPRA activity, and distal elements whose accessibility increases later in retinal development are enriched for CREs with silencer activity. We identified a set of potentially pleiotropic regulatory elements that convert from silencers to enhancers in retinas that lack a functional CRX effector domain. Our findings show that phenotypically distinct variants in different domains of CRX have partially overlapping effects on its cis-regulatory function, leading to misregulation of similar sets of enhancers while having a qualitatively different impact on silencers.

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.

RalB and the exocyst mediate the cellular starvation response by direct activation of autophagosome assembly.

The study of macroautophagy in mammalian cells has described induction, vesicle nucleation, and membrane elongation complexes as key signaling intermediates driving autophagosome biogenesis. How these components are recruited to nascent autophagosomes is poorly understood, and although much is known about signaling mechanisms that restrain autophagy, the nature of positive inductive signals that can promote autophagy remain cryptic. We find that the Ras-like small G protein, RalB, is localized to nascent autophagosomes and is activated on nutrient deprivation. RalB and its effector Exo84 are required for nutrient starvation-induced autophagocytosis, and RalB activation is sufficient to promote autophagosome formation. Through direct binding to Exo84, RalB induces the assembly of catalytically active ULK1 and Beclin1-VPS34 complexes on the exocyst, which are required for isolation membrane formation and maturation. Thus, RalB signaling is a primary adaptive response to nutrient limitation that directly engages autophagocytosis through mobilization of the core vesicle nucleation machinery.

Positive Selection Drives cis-regulatory Evolution Across the Threespine Stickleback Y Chromosome.

Allele-specific gene expression evolves rapidly on heteromorphic sex chromosomes. Over time, the accumulation of mutations on the Y chromosome leads to widespread loss of gametolog expression, relative to the X chromosome. It remains unclear if expression evolution on degrading Y chromosomes is primarily driven by mutations that accumulate through processes of selective interference, or if positive selection can also favor the down-regulation of coding regions on the Y chromosome that contain deleterious mutations. Identifying the relative rates of cis-regulatory sequence evolution across Y chromosomes has been challenging due to the limited number of reference assemblies. The threespine stickleback (Gasterosteus aculeatus) Y chromosome is an excellent model to identify how regulatory mutations accumulate on Y chromosomes due to its intermediate state of divergence from the X chromosome. A large number of Y-linked gametologs still exist across 3 differently aged evolutionary strata to test these hypotheses. We found that putative enhancer regions on the Y chromosome exhibited elevated substitution rates and decreased polymorphism when compared to nonfunctional sites, like intergenic regions and synonymous sites. This suggests that many cis-regulatory regions are under positive selection on the Y chromosome. This divergence was correlated with X-biased gametolog expression, indicating the loss of expression from the Y chromosome may be favored by selection. Our findings provide evidence that Y-linked cis-regulatory regions exhibit signs of positive selection quickly after the suppression of recombination and allow comparisons with recent theoretical models that suggest the rapid divergence of regulatory regions may be favored to mask deleterious mutations on the Y chromosome.

The evolution of gene regulation on sex chromosomes.

Sex chromosomes have evolved repeatedly across the tree of life. Most work has focused on the loss of coding regions from sex-limited chromosomes through the accumulation of deleterious mutations. By comparison, less is known about how the regulatory landscape evolves. We review theories of how regulatory landscapes evolve on sex chromosomes and the overall impact they have on gametolog expression. We integrate empirical studies on sex chromosomes with theoretical work to synthesize how regulatory evolution could occur on sex chromosomes. Recent findings have revealed that downregulation of ancestral alleles is probably widespread on Y chromosomes and that regulatory evolution plays a key role in the evolution of sex chromosomes.

Transcription factor interactions explain the context-dependent activity of CRX binding sites.

The effects of transcription factor binding sites (TFBSs) on the activity of a cis-regulatory element (CRE) depend on the local sequence context. In rod photoreceptors, binding sites for the transcription factor (TF) Cone-rod homeobox (CRX) occur in both enhancers and silencers, but the sequence context that determines whether CRX binding sites contribute to activation or repression of transcription is not understood. To investigate the context-dependent activity of CRX sites, we fit neural network-based models to the activities of synthetic CREs composed of photoreceptor TFBSs. The models revealed that CRX binding sites consistently make positive, independent contributions to CRE activity, while negative homotypic interactions between sites cause CREs composed of multiple CRX sites to function as silencers. The effects of negative homotypic interactions can be overcome by the presence of other TFBSs that either interact cooperatively with CRX sites or make independent positive contributions to activity. The context-dependent activity of CRX sites is thus determined by the balance between positive heterotypic interactions, independent contributions of TFBSs, and negative homotypic interactions. Our findings explain observed patterns of activity among genomic CRX-bound enhancers and silencers, and suggest that enhancers may require diverse TFBSs to overcome negative homotypic interactions between TFBSs.

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.

Long-read RNA sequencing reveals widespread sex-specific alternative splicing in threespine stickleback fish.

Alternate isoforms are important contributors to phenotypic diversity across eukaryotes. Although short-read RNA-sequencing has increased our understanding of isoform diversity, it is challenging to accurately detect full-length transcripts, preventing the identification of many alternate isoforms. Long-read sequencing technologies have made it possible to sequence full-length alternative transcripts, accurately characterizing alternative splicing events, alternate transcription start and end sites, and differences in UTR regions. Here, we use Pacific Biosciences (PacBio) long-read RNA-sequencing (Iso-Seq) to examine the transcriptomes of five organs in threespine stickleback fish (Gasterosteus aculeatus), a widely used genetic model species. The threespine stickleback fish has a refined genome assembly in which gene annotations are based on short-read RNA sequencing and predictions from coding sequence of other species. This suggests some of the existing annotations may be inaccurate or alternative transcripts may not be fully characterized. Using Iso-Seq we detected thousands of novel isoforms, indicating many isoforms are absent in the current Ensembl gene annotations. In addition, we refined many of the existing annotations within the genome. We noted many improperly positioned transcription start sites that were refined with long-read sequencing. The Iso-Seq-predicted transcription start sites were more accurate and verified through ATAC-seq. We also detected many alternative splicing events between sexes and across organs. We found a substantial number of genes in both somatic and gonadal samples that had sex-specific isoforms. Our study highlights the power of long-read sequencing to study the complexity of transcriptomes, greatly improving genomic resources for the threespine stickleback fish.

Meiotic pairing and double-strand break formation along the heteromorphic threespine stickleback sex chromosomes.

Double-strand break repair during meiosis is normally achieved using the homologous chromosome as a repair template. Heteromorphic sex chromosomes share little sequence homology, presenting unique challenges to the repair of double-strand breaks. Our understanding of how heteromorphic sex chromosomes behave during meiosis has been focused on ancient sex chromosomes, where the X and Y differ markedly in overall structure and gene content. It remains unclear how more recently evolved sex chromosomes that share considerably more sequence homology with one another pair and form double-strand breaks. One possibility is barriers to pairing evolve rapidly. Alternatively, recently evolved sex chromosomes may exhibit pairing and double-strand break repair that more closely resembles that of their autosomal ancestors. Here, we use the recently evolved X and Y chromosomes of the threespine stickleback fish (Gasterosteus aculeatus) to study patterns of pairing and double-stranded break formation using molecular cytogenetics. We found that the sex chromosomes of threespine stickleback fish did not pair exclusively in the pseudoautosomal region. Instead, the chromosomes fully paired in a non-homologous fashion. To achieve this, the X chromosome underwent synaptic adjustment during pachytene to match the axis length of the Y chromosome. Double-strand break formation and repair rate also matched that of the autosomes. Our results highlight that recently evolved sex chromosomes exhibit meiotic behavior that is reminiscent of autosomes and argues for further work to identify the homologous templates that are used to repair double-strand breaks on the X and Y chromosomes.

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.

Disrupted Gene Networks in Subfertile Hybrid House Mice.

The Dobzhansky-Muller (DM) model provides a widely accepted mechanism for the evolution of reproductive isolation: incompatible substitutions disrupt interactions between genes. To date, few candidate incompatibility genes have been identified, leaving the genes driving speciation mostly uncharacterized. The importance of interactions in the DM model suggests that gene coexpression networks provide a powerful framework to understand disrupted pathways associated with postzygotic isolation. Here, we perform weighted gene coexpression network analysis to infer gene interactions in hybrids of two recently diverged European house mouse subspecies, Mus mus domesticus and M. m. musculus, which commonly show hybrid male sterility or subfertility. We use genome-wide testis expression data from 467 hybrid mice from two mapping populations: F2s from a laboratory cross between wild-derived pure subspecies strains and offspring of natural hybrids captured in the Central Europe hybrid zone. This large data set enabled us to build a robust consensus network using hybrid males with fertile phenotypes. We identify several expression modules, or groups of coexpressed genes, that are disrupted in subfertile hybrids, including modules functionally enriched for spermatogenesis, cilium and sperm flagellum organization, chromosome organization, and DNA repair, and including genes expressed in spermatogonia, spermatocytes, and spermatids. Our network-based approach enabled us to hone in on specific hub genes likely to be influencing module-wide gene expression and hence potentially driving large-effect DM incompatibilities. A disproportionate number of hub genes lie within sterility loci identified previously in the hybrid zone mapping population and represent promising candidate barrier genes and targets for future functional analysis.

Shaping the p53 response with nucleoporins.

In this issue, Singer et al. (2012) reveal that the nucleoporin Nup98 supports adaptation to genotoxic stress by protecting specific p53-induced mRNAs from exosome-dependent degradation, suggesting that wild-type Nup98 may possess tumor suppressor function.

Regulating, Measuring, and Modeling the Viscoelasticity of Bacterial Biofilms

Biofilms occur in a broad range of environments under heterogeneous physicochemical conditions, such as in bioremediation plants, on surfaces of biomedical implants, and in the lungs of cystic fibrosis patients. In these scenarios, biofilms are subjected to shear forces, but the mechanical integrity of these aggregates often prevents their disruption or dispersal. Biofilms' physical robustness is the result of the multiple biopolymers secreted by constituent microbial cells which are also responsible for numerous biological functions. A better understanding of the role of these biopolymers and their response to dynamic forces is therefore crucial for understanding the interplay between biofilm structure and function. In this paper, we review experimental techniques in rheology, which help quantify the viscoelasticity of biofilms, and modeling approaches from soft matter physics that can assist our understanding of the rheological properties. We describe how these methods could be combined with synthetic biology approaches to control and investigate the effects of secreted polymers on the physical properties of biofilms. We argue that without an integrated approach of the three disciplines, the links between genetics, composition, and interaction of matrix biopolymers and the viscoelastic properties of biofilms will be much harder to uncover.

Selective Cytotoxicity of the NAMPT Inhibitor FK866 Toward Gastric Cancer Cells With Markers of the Epithelial-Mesenchymal Transition, Due to Loss of NAPRT.

BACKGROUND & AIMS: Markers of the epithelial-to-mesenchymal transition (EMT) in gastric tumor tissues are associated with poor patient outcomes. We performed a screen to identify pharmacologic compounds that kill gastric cancer cells with EMT-associated gene expression patterns and investigate their mechanisms. METHODS: We identified 29 gastric cancer cell lines with a gene expression signature previously associated with an EMT subtype, based on data from RNA sequence analyses, and confirmed the mesenchymal phenotypes of 7 lines (Hs746T, SNU1750, MKN1, SK4, SNU484, SNU668, and YCC11), based on invasive activity and protein markers. We screened 1,345 compounds for their ability to kill cells with the EMT signature compared with cell lines without this pattern. We tested the effects of identified compounds in BALB/c nude mice bearing GA077 tumors; mice were given intraperitoneal injections of the compound or vehicle (control) twice daily for 24 days and tumor growth was monitored. Proteins associated with the toxicity of the compounds were overexpressed in MKN1 and SNU484 cells or knocked down in MKN45 and SNU719 using small interfering RNAs. We performed immunohistochemical analyses of 942 gastric cancer tissues and investigated associations between EMT markers and protein expression patterns. RESULTS: The nicotinamide phosphoribosyltransferase inhibitor FK866 killed 6 of 7 gastric cancer cell lines with EMT-associated gene expression signatures but not gastric cancer cells without this signature. The 6 EMT-subtype gastric cell lines expressed significantly low levels of nicotinic acid phosphoribosyltransferase (NAPRT), which makes the cells hypersensitive to nicotinamide phosphoribosyltransferase inhibition. Gastric cell lines that expressed higher levels of NAPRT, regardless of EMT markers, were sensitized to FK866 after knockdown of NAPRT, whereas overexpression of NAPRT in deficient EMT cell lines protected them from FK866-mediated toxicity. Administration of FK866 to nude mice with tumors grown from GA077 cells (human gastric cancer tumors of the EMT subtype) led to tumor regression in 2 weeks; FK866 did not affect tumors grown from MKN45 cells without the EMT expression signature. Loss of NAPRT might promote the EMT, because it stabilizes ß-catenin. We correlated the EMT gene expression signature with lower levels of NAPRT in 942 gastric tumors from patients; we also found lower levels of NAPRT mRNA in colorectal, pancreatic, and lung adenocarcinoma tissues with the EMT gene expression signature. CONCLUSIONS: FK866 selectively kills gastric cancer cells with an EMT gene expression signature by inhibiting nicotinamide phosphoribosyltransferase in cells with NAPRT deficiency. Loss of NAPRT expression, frequently through promoter hypermethylation, is observed in many gastric tumors of the EMT subtype. FK866 might be used to treat patients with tumors of this subtype.

TBK1 directly engages Akt/PKB survival signaling to support oncogenic transformation.

The innate immune-signaling kinase, TBK1, couples pathogen surveillance to induction of host defense mechanisms. Pathological activation of TBK1 in cancer can overcome programmed cell death cues, enabling cells to survive oncogenic stress. The mechanistic basis of TBK1 prosurvival signaling, however, has been enigmatic. Here, we show that TBK1 directly activates AKT by phosphorylation of the canonical activation loop and hydrophobic motif sites independently of PDK1 and mTORC2. Upon mitogen stimulation, triggering of the innate immune response, re-exposure to glucose, or oncogene activation, TBK1 is recruited to the exocyst, where it activates AKT. In cells lacking TBK1, insulin activates AKT normally, but AKT activation by exocyst-dependent mechanisms is impaired. Discovery and characterization of a 6-aminopyrazolopyrimidine derivative, as a selective low-nanomolar TBK1 inhibitor, indicates that this regulatory arm can be pharmacologically perturbed independently of canonical PI3K/PDK1 signaling. Thus, AKT is a direct TBK1 substrate that connects TBK1 to prosurvival signaling.