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Abstract Chemical probes interrogate disease mechanisms at the molecular level by linking genetic changes to observable traits. However, comprehensive chemical screens in diverse biological models are impractical. To address this challenge, we develop ChemProbe, a model that predicts cellular sensitivity to hundreds of molecular probes and drugs by learning to combine transcriptomes and chemical structures. Using ChemProbe, we infer the chemical sensitivity of cancer cell lines and tumor samples and analyze how the model makes predictions. We retrospectively evaluate drug response predictions for precision breast cancer treatment and prospectively validate chemical sensitivity predictions in new cellular models, including a genetically modified cell line. Our model interpretation analysis identifies transcriptome features reflecting compound targets and protein network modules, identifying genes that drive ferroptosis. ChemProbe is an interpretable in silico screening tool that allows researchers to measure cellular response to diverse compounds, facilitating research into molecular mechanisms of chemical sensitivity.

Abstract In our cells, a limited number of RNA binding proteins (RBPs) are responsible for all aspects of RNA metabolism across the entire transcriptome. To accomplish this, RBPs form regulatory units that act on specific target regulons. However, the landscape of RBP combinatorial interactions remains poorly explored. Here, we perform a systematic annotation of RBP combinatorial interactions via multimodal data integration. We build a large-scale map of RBP protein neighborhoods by generating in vivo proximity-dependent biotinylation datasets of 50 human RBPs. In parallel, we use CRISPR interference with single-cell readout to capture transcriptomic changes upon RBP knockdowns. By combining these physical and functional interaction readouts, along with the atlas of RBP mRNA targets from eCLIP assays, we generate an integrated map of functional RBP interactions. We then use this map to match RBPs to their context-specific functions and validate the predicted functions biochemically for four RBPs. This study provides a detailed map of RBP interactions and deconvolves them into distinct regulatory modules with annotated functions and target regulons. This multimodal and integrative framework provides a principled approach for studying post-transcriptional regulatory processes and enriches our understanding of their underlying mechanisms.

Abstract Targeted therapy is effective in many tumor types including lung cancer, the leading cause of cancer mortality. Paradigm defining examples are targeted therapies directed against non-small cell lung cancer (NSCLC) subtypes with oncogenic alterations in EGFR, ALK and KRAS. The success of targeted therapy is limited by drug-tolerant persister cells (DTPs) which withstand and adapt to treatment and comprise the residual disease state that is typical during treatment with clinical targeted therapies. Here, we integrate studies in patient-derived and immunocompetent lung cancer models and clinical specimens obtained from patients on targeted therapy to uncover a focal adhesion kinase (FAK)-YAP signaling axis that promotes residual disease during oncogenic EGFR-, ALK-, and KRAS-targeted therapies. FAK-YAP signaling inhibition combined with the primary targeted therapy suppressed residual drug-tolerant cells and enhanced tumor responses. This study unveils a FAK-YAP signaling module that promotes residual disease in lung cancer and mechanism-based therapeutic strategies to improve tumor response.

Summary: Microsatellite instability-high (MSI-H) tumors are malignant tumors that, despite harboring a high mutational burden, often have intact TP53. One of the most frequent mutations in MSI-H tumors is a frameshift mutation in RPL22, a ribosomal protein. Here, we identified RPL22 as a modulator of MDM4 splicing through an alternative splicing switch in exon 6. RPL22 loss increases MDM4 exon 6 inclusion and cell proliferation and augments resistance to the MDM inhibitor Nutlin-3a. RPL22 represses the expression of its paralog, RPL22L1, by mediating the splicing of a cryptic exon corresponding to a truncated transcript. Therefore, damaging mutations in RPL22 drive oncogenic MDM4 induction and reveal a common splicing circuit in MSI-H tumors that may inform therapeutic targeting of the MDM4-p53 axis and oncogenic RPL22L1 induction.

Abstract Background Despite advances in early detection and therapies, cancer is still one of the most common causes of death worldwide. Since each tumor is unique, there is a need to implement personalized care and develop robust tools for monitoring treatment response to assess drug efficacy and prevent disease relapse. Main body Recent developments in liquid biopsies have enabled real-time noninvasive monitoring of tumor burden through the detection of molecules shed by tumors in the blood. These molecules include circulating tumor nucleic acids (ctNAs), comprising cell-free DNA or RNA molecules passively and/or actively released from tumor cells. Often highlighted for their diagnostic, predictive, and prognostic potential, these biomarkers possess valuable information about tumor characteristics and evolution. While circulating tumor DNA (ctDNA) has been in the spotlight for the last decade, less is known about circulating tumor RNA (ctRNA). There are unanswered questions about why some tumors shed high amounts of ctNAs while others have undetectable levels. Also, there are gaps in our understanding of associations between tumor evolution and ctNA characteristics and shedding kinetics. In this review, we summarize current knowledge about ctNA biology and release mechanisms and put this information into the context of tumor evolution and clinical utility. Conclusions A deeper understanding of the biology of ctDNA and ctRNA may inform the use of liquid biopsies in personalized medicine to improve cancer patient outcomes.

IntroductionDrug resistance is a major obstacle in cancer treatment and can involve a variety of different factors. Identifying effective therapies for drug resistant tumors is integral for improving patient outcomes.MethodsIn this study, we applied a computational drug repositioning approach to identify potential agents to sensitize primary drug resistant breast cancers. We extracted drug resistance profiles from the I-SPY 2 TRIAL, a neoadjuvant trial for early stage breast cancer, by comparing gene expression profiles of responder and non-responder patients stratified into treatments within HR/HER2 receptor subtypes, yielding 17 treatment-subtype pairs. We then used a rank-based pattern-matching strategy to identify compounds in the Connectivity Map, a database of cell line derived drug perturbation profiles, that can reverse these signatures in a breast cancer cell line. We hypothesize that reversing these drug resistance signatures will sensitize tumors to treatment and prolong survival.ResultsWe found that few individual genes are shared among the drug resistance profiles of different agents. At the pathway level, however, we found enrichment of immune pathways in the responders in 8 treatments within the HR+HER2+, HR+HER2-, and HR-HER2- receptor subtypes. We also found enrichment of estrogen response pathways in the non-responders in 10 treatments primarily within the hormone receptor positive subtypes. Although most of our drug predictions are unique to treatment arms and receptor subtypes, our drug repositioning pipeline identified the estrogen receptor antagonist fulvestrant as a compound that can potentially reverse resistance across 13/17 of the treatments and receptor subtypes including HR+ and triple negative. While fulvestrant showed limited efficacy when tested in a panel of 5 paclitaxel resistant breast cancer cell lines, it did increase drug response in combination with paclitaxel in HCC-1937, a triple negative breast cancer cell line.ConclusionWe applied a computational drug repurposing approach to identify potential agents to sensitize drug resistant breast cancers in the I-SPY 2 TRIAL. We identified fulvestrant as a potential drug hit and showed that it increased response in a paclitaxel-resistant triple negative breast cancer cell line, HCC-1937, when treated in combination with paclitaxel.

Abstract The extracellular activity of Plasminogen activator inhibitor-1 (PAI-1) is well described, acting as an inhibitor of tissue plasminogen activator and urokinase-type plasminogen activator, impacting fibrinolysis. Recent studies have revealed a pro-tumorigenic role of PAI-1 in human cancers, via the regulation of angiogenesis and tumor cell survival. In this study, immunohistochemical staining of 939 human bladder cancer specimens showed that PAI-1 expression levels correlated with tumor grade, tumor stage and overall survival. The typical subcellular localization of PAI-1 is cytoplasmic, but in approximately a quarter of the cases, PAI-1 was observed to be localized to both the tumor cell cytoplasm and the nucleus. To investigate the potential function of nuclear PAI-1 in tumor biology we applied chromatin immunoprecipitation (ChIP)-sequencing, gene expression profiling, and rapid immunoprecipitation mass spectrometry to a pair of bladder cancer cell lines. ChIP-sequencing revealed that PAI-1 can bind DNA at distal intergenic regions, suggesting a role as a transcriptional coregulator. The downregulation of PAI-1 in bladder cancer cell lines caused the upregulation of numerous genes, and the integration of ChIP-sequence and RNA-sequence data identified 57 candidate genes subject to PAI-1 regulation. Taken together, the data suggest that nuclear PAI-1 can influence gene expression programs and support malignancy.

Abstract Cancer metastasis is the primary cause of the high mortality rate among human cancers. Efforts to identify therapeutic agents targeting cancer metastasis frequently fail to demonstrate efficacy in clinical trials despite strong preclinical evidence. Until recently, most preclinical studies used mouse models to evaluate anti-metastatic agents. Mouse models are time-consuming and expensive. In addition, an important drawback is that mouse models inadequately model the early stages of metastasis which plausibly leads to the poor correlation with clinical outcomes. Here, we report an in vivo model based on xenografted zebrafish embryos where we select for progressively invasive subpopulations of MDA-MB-231 breast cancer cells. A subpopulation analogous to circulating tumor cells found in human cancers was selected by injection of MDA-MB-231 cells into the yolk sacs of 2 days post-fertilized zebrafish embryos and selecting cells that migrated to the tail. The selected subpopulation derived from MDA-MB-231 cells were increasingly invasive in zebrafish. Isolation of these subpopulations and propagation in vitro revealed morphological changes consistent with activation of an epithelial-mesenchymal transition program. Differential gene analysis and knockdown of genes identified gene-candidates (DDIT4, MT1X, CTSD, and SERPINE1) as potential targets for anti-metastasis therapeutics. Furthermore, RNA-splicing analysis reinforced the importance of BIRC5 splice variants in breast cancer metastasis. This is the first report using zebrafish to isolate and expand progressively invasive populations of human cancer cells. The model has potential applications in understanding the metastatic process, identification and/or development of therapeutics that specifically target metastatic cells and formulating personalized treatment strategies for individual cancer patients.

Obesity is linked to cancer risk in post-menopausal breast cancer. At the molecular level this is governed by cellular adaption to palmitic acid through epigenetic activation of a C/EBPB-dependent transcriptional network that drives tumor formation.

Abstract Background Advances in cancer biology are increasingly dependent on integration of heterogeneous datasets. Large-scale efforts have systematically mapped many aspects of cancer cell biology; however, it remains challenging for individual scientists to effectively integrate and understand this data. Results We have developed a new data retrieval and indexing framework that allows us to integrate publicly available data from different sources and to combine publicly available data with new or bespoke datasets. Our approach, which we have named the cancer data integrator (CanDI), is straightforward to implement, is well documented, and is continuously updated which should enable individual users to take full advantage of efforts to map cancer cell biology. We show that CanDI empowered testable hypotheses of new synthetic lethal gene pairs, genes associated with sex disparity, and immunotherapy targets in cancer. Conclusions CanDI provides a flexible approach for large-scale data integration in cancer research enabling rapid generation of hypotheses. The CanDI data integrator is available at https://github.com/GilbertLabUCSF/CanDI .

Kras is frequently mutated in lung cancer and two isoforms are generated via alternative splicing. Here, the authors show that the two isoforms have divergent roles in cancer stem cells and the main tumour cell population, which are regulated by hypoxia and endoplasmic reticulum stress.

Summary: The major cap-binding protein eukaryotic translation initiation factor 4E (eIF4E), an ancient protein required for translation of all eukaryotic genomes, is a surprising yet potent oncogenic driver. The genetic interactions that maintain the oncogenic activity of this key translation factor remain unknown. In this study, we carry out a genome-wide CRISPRi screen wherein we identify more than 600 genetic interactions that sustain eIF4E oncogenic activity. Our data show that eIF4E controls the translation of Tfeb, a key executer of the autophagy response. This autophagy survival response is triggered by mitochondrial proteotoxic stress, which allows cancer cell survival. Our screen also reveals a functional interaction between eIF4E and a single anti-apoptotic factor, Bcl-xL, in tumor growth. Furthermore, we show that eIF4E and the exon-junction complex (EJC), which is involved in many steps of RNA metabolism, interact to control the migratory properties of cancer cells. Overall, we uncover several cancer-specific vulnerabilities that provide further resolution of the cancer translatome.

Our understanding of the beads-on-a-string arrangement of nucleosomes has been built largely on high-resolution sequence-agnostic imaging methods and sequence-resolved bulk biochemical techniques. To bridge the divide between these approaches, we present the single-molecule adenine methylated oligonucleosome sequencing assay (SAMOSA). SAMOSA is a high-throughput single-molecule sequencing method that combines adenine methyltransferase footprinting and single-molecule real-time DNA sequencing to natively and nondestructively measure nucleosome positions on individual chromatin fibres. SAMOSA data allows unbiased classification of single-molecular 'states' of nucleosome occupancy on individual chromatin fibres. We leverage this to estimate nucleosome regularity and spacing on single chromatin fibres genome-wide, at predicted transcription factor binding motifs, and across human epigenomic domains. Our analyses suggest that chromatin is comprised of both regular and irregular single-molecular oligonucleosome patterns that differ subtly in their relative abundance across epigenomic domains. This irregularity is particularly striking in constitutive heterochromatin, which has typically been viewed as a conformationally static entity. Our proof-of-concept study provides a powerful new methodology for studying nucleosome organization at a previously intractable resolution and offers up new avenues for modeling and visualizing higher order chromatin structure.

SARS-COV-2 has roused the scientific community with a call to action to combat the growing pandemic. At the time of this writing, there are as yet no novel antiviral agents or approved vaccines available for deployment as a frontline defense. Understanding the pathobiology of COVID-19 could aid scientists in their discovery of potent antivirals by elucidating unexplored viral pathways. One method for accomplishing this is the leveraging of computational methods to discover new candidate drugs and vaccines in silico. In the last decade, machine learning-based models, trained on specific biomolecules, have offered inexpensive and rapid implementation methods for the discovery of effective viral therapies. Given a target biomolecule, these models are capable of predicting inhibitor candidates in a structural-based manner. If enough data are presented to a model, it can aid the search for a drug or vaccine candidate by identifying patterns within the data. In this review, we focus on the recent advances of COVID-19 drug and vaccine development using artificial intelligence and the potential of intelligent training for the discovery of COVID-19 therapeutics. To facilitate applications of deep learning for SARS-COV-2, we highlight multiple molecular targets of COVID-19, inhibition of which may increase patient survival. Moreover, we present CoronaDB-AI, a dataset of compounds, peptides, and epitopes discovered either in silico or in vitro that can be potentially used for training models in order to extract COVID-19 treatment. The information and datasets provided in this review can be used to train deep learning-based models and accelerate the discovery of effective viral therapies.

Colorectal cancer (CRC) is a major cause of human death. Mortality is primarily due to metastatic organ colonization, with the liver being the main organ affected. We modeled metastatic CRC (mCRC) liver colonization using patient-derived primary and metastatic tumor xenografts (PDX). Such PDX modeling predicted patient survival outcomes. In vivo selection of multiple PDXs for enhanced metastatic colonization capacity upregulated the gluconeogenic enzyme PCK1, which enhanced liver metastatic growth by driving pyrimidine nucleotide biosynthesis under hypoxia. Consistently, highly metastatic tumors upregulated multiple pyrimidine biosynthesis intermediary metabolites. Therapeutic inhibition of the pyrimidine biosynthetic enzyme DHODH with leflunomide substantially impaired CRC liver metastatic colonization and hypoxic growth. Our findings provide a potential mechanistic basis for the epidemiologic association of anti-gluconeogenic drugs with improved CRC metastasis outcomes, reveal the exploitation of a gluconeogenesis enzyme for pyrimidine biosynthesis under hypoxia, and implicate DHODH and PCK1 as metabolic therapeutic targets in CRC metastatic progression.

“mRNA abundance is determined by the rates of transcription and decay. Here, the authors propose a method for estimating the rate of differential mRNA decay from RNA-seq data and model mRNA stability in the brain, suggesting a link between mRNA stability and Alzheimer’s disease.”

Phenotypic and genetic intra-tumor heterogeneity have an important role in cancer progression and therapeutic resistance. Here, the authors show that phenotypically variable tumor subpopulations exhibit higher metastatic potential and display enhanced intra-clonal transcriptomic variability, likely promoted by deregulated spliceosome activity.

Accurate and precise annotation of 3′ UTRs is critical for understanding how mRNAs are regulated by microRNAs (miRNAs) and RNA-binding proteins (RBPs). Here, we describe a method, poly(A) binding protein-mediated mRNA 3′ end retrieval by crosslinking immunoprecipitation (PAPERCLIP), that shows high specificity for mRNA 3′ ends and compares favorably with existing 3′ end mapping methods. PAPERCLIP uncovers a previously unrecognized role of CstF64/64tau in promoting the usage of a selected group of non-canonical poly(A) sites, the majority of which contain a downstream GUKKU motif. Furthermore, in the mouse brain, PAPERCLIP discovers extended 3′ UTR sequences harboring functional miRNA binding sites and reveals developmentally regulated APA shifts, including one in Atp2b2 that is evolutionarily conserved in humans and results in the gain of a functional binding site of miR-137. PAPERCLIP provides a powerful tool to decipher post-transcriptional regulation of mRNAs through APA in vivo.

Background Preterm birth is a main determinant of neonatal mortality and morbidity and a major contributor to the overall mortality and burden of disease. However, research of the preterm birth is hindered by the imprecise definition of the clinical phenotype and complexity of the molecular phenotype due to multiple pregnancy tissue types and molecular processes that may contribute to the preterm birth. Here we comprehensively evaluate the mRNA transcriptome that characterizes preterm and term labor in tissues comprising the pregnancy using precisely phenotyped samples. The four complementary phenotypes together provide comprehensive insight into preterm and term parturition. Methods Samples of maternal blood, chorion, amnion, placenta, decidua, fetal blood, and myometrium from the uterine fundus and lower segment (n = 183) were obtained during cesarean delivery from women with four complementary phenotypes: delivering preterm with (PL) and without labor (PNL), term with (TL) and without labor (TNL). Enrolled were 35 pregnant women with four precisely and prospectively defined phenotypes: PL (n = 8), PNL (n = 10), TL (n = 7) and TNL (n = 10). Gene expression data were analyzed using shrunken centroid analysis to identify a minimal set of genes that uniquely characterizes each of the four phenotypes. Expression profiles of 73 genes and non-coding RNA sequences uniquely identified each of the four phenotypes. The shrunken centroid analysis and 10 times 10-fold cross-validation was also used to minimize false positive finings and overfitting. Identified were the pathways and molecular processes associated with and the cis-regulatory elements in gene’s 5′ promoter or 3′-UTR regions of the set of genes which expression uniquely characterized the four phenotypes. Results The largest differences in gene expression among the four groups occurred at maternal fetal interface in decidua, chorion and amnion. The gene expression profiles showed suppression of chemokines expression in TNL, withdrawal of this suppression in TL, activation of multiple pathways of inflammation in PL, and an immune rejection profile in PNL. The genes constituting expression signatures showed over-representation of three putative regulatory elements in their 5′and 3′ UTR regions. Conclusions The results suggest that pregnancy is maintained by downregulation of chemokines at the maternal-fetal interface. Withdrawal of this downregulation results in the term birth and its overriding by the activation of multiple pathways of the immune system in the preterm birth. Complications of the pregnancy associated with impairment of placental function, which necessitated premature delivery of the fetus in the absence of labor, show gene expression patterns associated with immune rejection.

Posttranscriptional regulatory programs governing diverse aspects of RNA biology remain largely uncharacterized. Understanding the functional roles of RNA cis-regulatory elements is essential for decoding complex programs that underlie the dynamic regulation of transcript stability, splicing, localization, and translation. Here, we describe a combined experimental/computational technology to reveal a catalog of functional regulatory elements embedded in 3′ UTRs of human transcripts. We used a bidirectional reporter system coupled with flow cytometry and high-throughput sequencing to measure the effect of short, noncoding, vertebrate-conserved RNA sequences on transcript stability and translation. Information-theoretic motif analysis of the resulting sequence-to-gene-expression mapping revealed linear and structural RNA cis-regulatory elements that positively and negatively modulate the posttranscriptional fates of human transcripts. This combined experimental/computational strategy can be used to systematically characterize the vast landscape of posttranscriptional regulatory elements controlling physiological and pathological cellular state transitions.

Microglia are resident immune cells of the CNS that are activated by infection, neuronal injury, and inflammation. Here, we utilize flow cytometry and deep RNA sequencing of acutely isolated spinal cord microglia to define their activation in vivo. Analysis of resting microglia identified 29 genes that distinguish microglia from other CNS cells and peripheral macrophages/monocytes. We then analyzed molecular changes in microglia during neurodegenerative disease activation using the SOD1G93A mouse model of amyotrophic lateral sclerosis (ALS). We found that SOD1G93A microglia are not derived from infiltrating monocytes, and that both potentially neuroprotective and toxic factors, including Alzheimer’s disease genes, are concurrently upregulated. Mutant microglia differed from SOD1WT, lipopolysaccharide-activated microglia, and M1/M2 macrophages, defining an ALS-specific phenotype. Concurrent messenger RNA/fluorescence-activated cell sorting analysis revealed posttranscriptional regulation of microglia surface receptors and T cell-associated changes in the transcriptome. These results provide insights into microglia biology and establish a resource for future studies of neuroinflammation.

The original version of this Article contained an error in Figure 3, where panel d was inadvertently replaced with a duplicate of panel c during typesetting. Also, the legend of Figure 5f incorrectly read ‘310 AD patients (blue dots, r = –0.4) and 157 non-demented individuals (green dots, r = –0.1)’, and should have read ‘310 AD patients (blue dots, r = –0.1) and 157 non-demented individuals (green dots, r = –0.4)’. Both of these errors have now been corrected in both the PDF and HTML versions of the Article.

Abstract Understanding the genetic basis of adaptation is a central problem in biology. However, revealing the underlying molecular mechanisms has been challenging as changes in fitness may result from perturbations to many pathways, any of which may contribute relatively little. We have developed a combined experimental/computational framework to address this problem and used it to understand the genetic basis of ethanol tolerance in Escherichia coli. We used fitness profiling to measure the consequences of single‐locus perturbations in the context of ethanol exposure. A module‐level computational analysis was then used to reveal the organization of the contributing loci into cellular processes and regulatory pathways (e.g. osmoregulation and cell‐wall biogenesis) whose modifications significantly affect ethanol tolerance. Strikingly, we discovered that a dominant component of adaptation involves metabolic rewiring that boosts intracellular ethanol degradation and assimilation. Through phenotypic and metabolomic analysis of laboratory‐evolved ethanol‐tolerant strains, we investigated naturally accessible pathways of ethanol tolerance. Remarkably, these laboratory‐evolved strains, by and large, follow the same adaptive paths as inferred from our coarse‐grained search of the fitness landscape.

Regulatory networks allow organisms to match adaptive behavior to the complex and dynamic contingencies of their native habitats. Upon a sudden transition to a novel environment, the mismatch between the native behavior and the new niche provides selective pressure for adaptive evolution through mutations in elements that control gene expression. In the case of core components of cellular regulation and metabolism, with broad control over diverse biological processes, such mutations may have substantial pleiotropic consequences. Through extensive phenotypic analyses, we have characterized the systems-level consequences of one such mutation (rho*) in the global transcriptional terminator Rho of Escherichia coli. We find that a single amino acid change in Rho results in a massive change in the fitness landscape of the cell, with widely discrepant fitness consequences of identical single locus perturbations in rho* versus rho(WT) backgrounds. Our observations reveal the extent to which a single regulatory mutation can transform the entire fitness landscape of the cell, causing a massive change in the interpretation of individual mutations and altering the evolutionary trajectories which may be accessible to a bacterial population.

Microbial biofilms are a dominant feature of many human infections. However, developing effective strategies for controlling biofilms requires an understanding of the underlying biology well beyond what currently exists. Using a novel strategy, we have induced formation of a robust biofilm in Escherichia coli by utilizing an exogenous source of poly-N-acetylglucosamine (PNAG) polymer, a major virulence factor of many pathogens. Through microarray profiling of competitive selections, carried out in both transposon insertion and over-expression libraries, we have revealed the genetic basis of PNAG-based biofilm formation. Our observations reveal the dominance of electrostatic interactions between PNAG and surface structures such as lipopolysaccharides. We show that regulatory modulation of these surface structures has significant impact on biofilm formation behavior of the cell. Furthermore, the majority of clinical isolates which produced PNAG also showed the capacity to respond to the exogenously produced version of the polymer.