Multimodal mass spectrometry imaging identifies cell-type-specific metabolic and lipidomic variation in the mammalian liver.

Spatial single-cell omics provides a readout of biochemical processes. It is challenging to capture the transient lipidome/metabolome from cells in a native tissue environment. We employed water gas cluster ion beam secondary ion mass spectrometry imaging ([H2O]n>28K-GCIB-SIMS) at ≤3 µm resolution using a cryogenic imaging workflow. This allowed multiple biomolecular imaging modes on the near-native-state liver at single-cell resolution. Our workflow utilizes desorption electrospray ionization (DESI) to build a reference map of metabolic heterogeneity and zonation across liver functional units at tissue level. Cryogenic dual-SIMS integrated metabolomics, lipidomics, and proteomics in the same liver lobules at single-cell level, characterizing the cellular landscape and metabolic states in different cell types. Lipids and metabolites classified liver metabolic zones, cell types and subtypes, highlighting the power of spatial multi-omics at high spatial resolution for understanding celluar and biomolecular organizations in the mammalian liver.

Subtype-selective prenylated isoflavonoids disrupt regulatory drivers of MYCN-amplified cancers.

Transcription factors have proven difficult to target with small molecules because they lack pockets necessary for potent binding. Disruption of protein expression can suppress targets and enable therapeutic intervention. To this end, we developed a drug discovery workflow that incorporates cell-line-selective screening and high-throughput expression profiling followed by regulatory network analysis to identify compounds that suppress regulatory drivers of disease. Applying this approach to neuroblastoma (NBL), we screened bioactive molecules in cell lines representing its MYC-dependent (MYCNA) and mesenchymal (MES) subtypes to identify selective compounds, followed by PLATESeq profiling of treated cells. This revealed compounds that disrupt a sub-network of MYCNA-specific regulatory proteins, resulting in MYCN degradation in vivo. The top hit was isopomiferin, a prenylated isoflavonoid that inhibited casein kinase 2 (CK2) in cells. Isopomiferin and its structural analogs inhibited MYC and MYCN in NBL and lung cancer cells, highlighting the general MYC-inhibiting potential of this unique scaffold.

Spatial pharmacology using mass spectrometry imaging.

The emerging and powerful field of spatial pharmacology can map the spatial distribution of drugs and their metabolites, as well as their effects on endogenous biomolecules including metabolites, lipids, proteins, peptides, and glycans, without the need for labeling. This is enabled by mass spectrometry imaging (MSI) that provides previously inaccessible information in diverse phases of drug discovery and development. We provide a perspective on how MSI technologies and computational tools can be implemented to reveal quantitative spatial drug pharmacokinetics and toxicology, tissue subtyping, and associated biomarkers. We also highlight the emerging potential of comprehensive spatial pharmacology through integration of multimodal MSI data with other spatial technologies. Finally, we describe how to overcome challenges including improving reproducibility and compound annotation to generate robust conclusions that will improve drug discovery and development processes.

Organ Mapping Antibody Panels: a community resource for standardized multiplexed tissue imaging.

Multiplexed antibody-based imaging enables the detailed characterization of molecular and cellular organization in tissues. Advances in the field now allow high-parameter data collection (>60 targets); however, considerable expertise and capital are needed to construct the antibody panels employed by these methods. Organ mapping antibody panels are community-validated resources that save time and money, increase reproducibility, accelerate discovery and support the construction of a Human Reference Atlas.

Development of optimized drug-like small molecule inhibitors of the SARS-CoV-2 3CL protease for treatment of COVID-19.

The SARS-CoV-2 3CL protease is a critical drug target for small molecule COVID-19 therapy, given its likely druggability and essentiality in the viral maturation and replication cycle. Based on the conservation of 3CL protease substrate binding pockets across coronaviruses and using screening, we identified four structurally distinct lead compounds that inhibit SARS-CoV-2 3CL protease. After evaluation of their binding specificity, cellular antiviral potency, metabolic stability, and water solubility, we prioritized the GC376 scaffold as being optimal for optimization. We identified multiple drug-like compounds with <10 nM potency for inhibiting SARS-CoV-2 3CL and the ability to block SARS-CoV-2 replication in human cells, obtained co-crystal structures of the 3CL protease in complex with these compounds, and determined that they have pan-coronavirus activity. We selected one compound, termed coronastat, as an optimized lead and characterized it in pharmacokinetic and safety studies in vivo. Coronastat represents a new candidate for a small molecule protease inhibitor for the treatment of SARS-CoV-2 infection for eliminating pandemics involving coronaviruses.

Time Course of Changes in Sorafenib-Treated Hepatocellular Carcinoma Cells Suggests Involvement of Phospho-Regulated Signaling in Ferroptosis Induction.

Ferroptosis is a form of regulated, non-apoptotic cell death characterized by excessive lipid peroxidation that can be triggered by inhibition of the cystine-glutamate antiporter, system Xc- . Sorafenib, an FDA-approved multi-kinase inhibitor drug that is used for treatment of hepatocellular carcinoma (HCC), has been shown to induce ferroptosis. Protein phosphorylation changes upon sorafenib treatment have been previously reported in patient studies and in cell culture. However, early phosphorylation changes during induction of ferroptosis are not reported. This work highlights these changes through a time course from 7 to 60 min of sorafenib treatment in human (SKHep1) HCC cells. A total of 6170 unique phosphosites from 2381 phosphoproteins are quantified, and phosphorylation changes occur after as little as 30 min of sorafenib treatment. By 60 min, notable changes included phosphosites significantly changing on p53 (P04637), CAD protein (P27708), and proteins important for iron homeostasis, such as heavy chain ferritin (FTH1; P02794), heme oxygenase 1 (HMOX1; P09601), and PCBP1 (Q15365). Additional sites on proteins in key regulatory pathways are identified, including sites in ferroptosis-related proteins, indicating the likely involvement of phospho-regulated signaling during ferroptosis induction.

Transferrin Receptor Is a Specific Ferroptosis Marker.

Ferroptosis is a type of regulated cell death driven by the iron-dependent accumulation of oxidized polyunsaturated fatty acid-containing phospholipids. There is no reliable way to selectively stain ferroptotic cells in tissue sections to characterize the extent of ferroptosis in animal models or patient samples. We address this gap by immunizing mice with membranes from lymphoma cells treated with the ferroptosis inducer piperazine erastin and screening ∼4,750 of the resulting monoclonal antibodies generated for their ability to selectively detect cells undergoing ferroptosis. We find that one antibody, 3F3 ferroptotic membrane antibody (3F3-FMA), is effective as a selective ferroptosis-staining reagent. The antigen of 3F3-FMA is identified as the human transferrin receptor 1 protein (TfR1). We validate this finding with several additional anti-TfR1 antibodies and compare them to other potential ferroptosis-detecting reagents. We find that anti-TfR1 and anti-malondialdehyde adduct antibodies are effective at staining ferroptotic tumor cells in multiple cell culture and tissue contexts.

Quality of labor and birth care in Sindh Province, Pakistan: Findings from direct observations at health facilities.

This study presents data from the first observation of labor, childbirth and immediate newborn care in a clinical setting in Sindh, the second most populous province of Pakistan. Trained midwives observed 310 births at 126 district level referral facilities and primary health care facilities in 10 districts of Sindh where the USAID-funded Maternal Child Health Integrated Program (MCHIP) was implemented. The facility participation rate was 78%. The findings show that monitoring vital signs during the initial examination was conducted for less than one-in-ten women. Infection prevention practices were only observed for one-in-four women. Modesty was preserved for less than half of women. In spite of an absence of monitoring during the first and second stages of labor, providers augmented labor with oxytocin in two-thirds of births. To prevent post-partum hemorrhage, oxytocin was administered within a minute of birth in 51% of cases. Immediate drying of the baby was nearly universal and eight out of ten babies were wrapped in a dry towel. Newborn vital signs and the baby's weight were taken in one-in-ten cases. Breastfeeding was initiated during the first hour of birth in 18% of cases. A support-person was present during labor and birth for 90% of women. While quality of care is poor across all facilities, the provision of care at district-level referral facilities was even lower quality than at primary health care facilities. This is because dais or assistants without formal training provided labor, birth, and newborn care for 40% of deliveries during night shifts at referral facilities. This study found many examples of suboptimal practice by skilled birth attendants across all levels of health facilities. There remains an urgent need to improve quality of service provision among skilled birth attendants in Pakistan.

Transcription factor activating protein 4 is synthetically lethal and a master regulator of MYCN-amplified neuroblastoma.

Despite the identification of MYCN amplification as an adverse prognostic marker in neuroblastoma, MYCN inhibitors have yet to be developed. Here, by integrating evidence from a whole-genome shRNA library screen and the computational inference of master regulator proteins, we identify transcription factor activating protein 4 (TFAP4) as a critical effector of MYCN amplification in neuroblastoma, providing a novel synthetic lethal target. We demonstrate that TFAP4 is a direct target of MYCN in neuroblastoma cells, and that its expression and activity strongly negatively correlate with neuroblastoma patient survival. Silencing TFAP4 selectively inhibits MYCN-amplified neuroblastoma cell growth both in vitro and in vivo, in xenograft mouse models. Mechanistically, silencing TFAP4 induces neuroblastoma differentiation, as evidenced by increased neurite outgrowth and upregulation of neuronal markers. Taken together, our results demonstrate that TFAP4 is a key regulator of MYCN-amplified neuroblastoma and may represent a valuable novel therapeutic target.

Cross-Cohort Analysis Identifies a TEAD4-MYCN Positive Feedback Loop as the Core Regulatory Element of High-Risk Neuroblastoma.

High-risk neuroblastomas show a paucity of recurrent somatic mutations at diagnosis. As a result, the molecular basis for this aggressive phenotype remains elusive. Recent progress in regulatory network analysis helped us elucidate disease-driving mechanisms downstream of genomic alterations, including recurrent chromosomal alterations. Our analysis identified three molecular subtypes of high-risk neuroblastomas, consistent with chromosomal alterations, and identified subtype-specific master regulator proteins that were conserved across independent cohorts. A 10-protein transcriptional module-centered around a TEAD4-MYCN positive feedback loop-emerged as the regulatory driver of the high-risk subtype associated with MYCN amplification. Silencing of either gene collapsed MYCN-amplified (MYCNAmp) neuroblastoma transcriptional hallmarks and abrogated viability in vitro and in vivo Consistently, TEAD4 emerged as a robust prognostic marker of poor survival, with activity independent of the canonical Hippo pathway transcriptional coactivators YAP and TAZ. These results suggest novel therapeutic strategies for the large subset of MYCN-deregulated neuroblastomas.Significance: Despite progress in understanding of neuroblastoma genetics, little progress has been made toward personalized treatment. Here, we present a framework to determine the downstream effectors of the genetic alterations sustaining neuroblastoma subtypes, which can be easily extended to other tumor types. We show the critical effect of disrupting a 10-protein module centered around a YAP/TAZ-independent TEAD4-MYCN positive feedback loop in MYCNAmp neuroblastomas, nominating TEAD4 as a novel candidate for therapeutic intervention. Cancer Discov; 8(5); 582-99. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 517.

Evaluation of a 5-year cervical cancer prevention project in Indonesia: opportunities, issues, and challenges.

AIM: The Cervical and Breast Cancer Prevention (CECAP) Project sought to develop a national model for cervical cancer prevention in Indonesia based on visual inspection with acetic acid (VIA) to detect abnormal changes in the cervix. The purpose of this study was to evaluate a pilot project introducing VIA and cryotherapy in Indonesia and to identify lessons learned that could be applied to the national scale-up of cervical cancer prevention services. MATERIAL AND METHODS: Fifty-four months (July 2007 to December 2011) of service records at 17 health centers were abstracted and analyzed. The data were used to calculate the proportion of all women aged 30-50 who received VIA screening, the VIA-positive rate, the treatment rate, and the interval between screening and treatment. RESULTS: The 45 050 women screened during the project included 24.4% of the total female population in the target age group in the catchment area. Throughout the 5-year project, 83.1% of VIA-positive women sought cryotherapy. During the last 18 months of the project, after data collection tools were revised to more accurately reflect when cryotherapy was received, 13% of women were treated on the same day that they were screened. Among the 74% of women treated within 1 month of screening, the mean interval between screening and treatment was 7.2 days. CONCLUSION: As cervical cancer prevention services are scaled up throughout Indonesia, changes in the service delivery model and program management are needed to increase screening coverage, promote a single-visit approach, and ensure the quality of services.

Influencing women's actions on cervical cancer screening and treatment in Karawang District, Indonesia.

INTRODUCTION: The impact of cervical cancer prevention programs depends on persuading women to go for screening and, if needed, treatment. As part of an evaluation of a pilot project in Indonesia, qualitative research was conducted to explore the factors that influence women's decisions regarding screening and treatment and to generate practical recommendations to increase service coverage and reduce loss to follow up. METHODS: Research was conducted at 7 of the 17 public health centers in Karawang District that implemented the pilot project. Interviews and focus group discussions were held with 20 women, 20 husbands, 10 doctors, 18 midwives, 3 district health officials, and 16 advocacy team members. RESULTS: Free services and mobile outreach events encouraged women to go for screening, along with promotional efforts by community health workers, advocacy teams, and the mass media. Knowledge and perceptions were the most important barriers to screening: women were not aware of cervical cancer risks, did not know the disease was treatable, and were fatalistic. Factors facilitating treatment were social support from husbands, relatives, and friends and the encouragement and role modeling of health workers. Barriers to prompt treatment included limited access to services and the requirement for husband's consent for cryotherapy. CONCLUSION: As cervical cancer prevention services are scaled up throughout Indonesia, the findings suggest three strategies to expand screening coverage and ensure prompt treatment: strengthening community mobilization and advocacy activities, modifying the service delivery model to encourage a single visit approach to screening and treatment, and working to gain men's support.

An extensive microRNA-mediated network of RNA-RNA interactions regulates established oncogenic pathways in glioblastoma.

By analyzing gene expression data in glioblastoma in combination with matched microRNA profiles, we have uncovered a posttranscriptional regulation layer of surprising magnitude, comprising more than 248,000 microRNA (miR)-mediated interactions. These include ∼7,000 genes whose transcripts act as miR "sponges" and 148 genes that act through alternative, nonsponge interactions. Biochemical analyses in cell lines confirmed that this network regulates established drivers of tumor initiation and subtype implementation, including PTEN, PDGFRA, RB1, VEGFA, STAT3, and RUNX1, suggesting that these interactions mediate crosstalk between canonical oncogenic pathways. siRNA silencing of 13 miR-mediated PTEN regulators, whose locus deletions are predictive of PTEN expression variability, was sufficient to downregulate PTEN in a 3'UTR-dependent manner and to increase tumor cell growth rates. Thus, miR-mediated interactions provide a mechanistic, experimentally validated rationale for the loss of PTEN expression in a large number of glioma samples with an intact PTEN locus.

A human B-cell interactome identifies MYB and FOXM1 as master regulators of proliferation in germinal centers.

Assembly of a transcriptional and post-translational molecular interaction network in B cells, the human B-cell interactome (HBCI), reveals a hierarchical, transcriptional control module, where MYB and FOXM1 act as synergistic master regulators of proliferation in the germinal center (GC). Eighty percent of genes jointly regulated by these transcription factors are activated in the GC, including those encoding proteins in a complex regulating DNA pre-replication, replication, and mitosis. These results indicate that the HBCI analysis can be used for the identification of determinants of major human cell phenotypes and provides a paradigm of general applicability to normal and pathologic tissues.

A systems biology approach to transcription factor binding site prediction.

BACKGROUND: The elucidation of mammalian transcriptional regulatory networks holds great promise for both basic and translational research and remains one the greatest challenges to systems biology. Recent reverse engineering methods deduce regulatory interactions from large-scale mRNA expression profiles and cross-species conserved regulatory regions in DNA. Technical challenges faced by these methods include distinguishing between direct and indirect interactions, associating transcription regulators with predicted transcription factor binding sites (TFBSs), identifying non-linearly conserved binding sites across species, and providing realistic accuracy estimates. METHODOLOGY/PRINCIPAL FINDINGS: We address these challenges by closely integrating proven methods for regulatory network reverse engineering from mRNA expression data, linearly and non-linearly conserved regulatory region discovery, and TFBS evaluation and discovery. Using an extensive test set of high-likelihood interactions, which we collected in order to provide realistic prediction-accuracy estimates, we show that a careful integration of these methods leads to significant improvements in prediction accuracy. To verify our methods, we biochemically validated TFBS predictions made for both transcription factors (TFs) and co-factors; we validated binding site predictions made using a known E2F1 DNA-binding motif on E2F1 predicted promoter targets, known E2F1 and JUND motifs on JUND predicted promoter targets, and a de novo discovered motif for BCL6 on BCL6 predicted promoter targets. Finally, to demonstrate accuracy of prediction using an external dataset, we showed that sites matching predicted motifs for ZNF263 are significantly enriched in recent ZNF263 ChIP-seq data. CONCLUSIONS/SIGNIFICANCE: Using an integrative framework, we were able to address technical challenges faced by state of the art network reverse engineering methods, leading to significant improvement in direct-interaction detection and TFBS-discovery accuracy. We estimated the accuracy of our framework on a human B-cell specific test set, which may help guide future methodological development.

Genome-wide identification of post-translational modulators of transcription factor activity in human B cells.

The ability of a transcription factor (TF) to regulate its targets is modulated by a variety of genetic and epigenetic mechanisms, resulting in highly context-dependent regulatory networks. However, high-throughput methods for the identification of proteins that affect TF activity are still largely unavailable. Here we introduce an algorithm, modulator inference by network dynamics (MINDy), for the genome-wide identification of post-translational modulators of TF activity within a specific cellular context. When used to dissect the regulation of MYC activity in human B lymphocytes, the approach inferred novel modulators of MYC function, which act by distinct mechanisms, including protein turnover, transcription complex formation and selective enzyme recruitment. MINDy is generally applicable to study the post-translational modulation of mammalian TFs in any cellular context. As such it can be used to dissect context-specific signaling pathways and combinatorial transcriptional regulation.

Correlating measurements across samples improves accuracy of large-scale expression profile experiments.

Gene expression profiling technologies suffer from poor reproducibility across replicate experiments. However, when analyzing large datasets, probe-level expression profile correlation can help identify flawed probes and lead to the construction of truer probe sets with improved reproducibility. We describe methods to eliminate uninformative and flawed probes, account for dependence between probes, and address variability due to transcript-isoform mixtures. We test and validate our approach on Affymetrix microarrays and outline their future adaptation to other technologies.