Resonance assignments of cytochrome MtoD from the extracellular electron uptake pathway of sideroxydans lithotrophicus ES-1.

The contribution of Fe(II)-oxidizing bacteria to iron cycling in freshwater, groundwater, and marine environments has been widely recognized in recent years. These organisms perform extracellular electron transfer (EET), which constitutes the foundations of bioelectrochemical systems for the production of biofuels and bioenergy. It was proposed that the Gram-negative bacterium Sideroxydans lithotrophicus ES-1 oxidizes soluble ferrous Fe(II) at the surface of the cell and performs EET through the Mto redox pathway. This pathway is composed by the periplasmic monoheme cytochrome MtoD that is proposed to bridge electron transfer between the cell exterior and the cytoplasm. This makes its functional and structural characterization, as well as evaluating the interaction process with its physiological partners, essential for understanding the mechanisms underlying EET. Here, we report the complete assignment of the heme proton and carbon signals together with a near-complete assignment of 1H, 13C and 15N backbone and side chain resonances for the reduced, diamagnetic form of the protein. These data pave the way to identify and structurally map the molecular interaction regions between the cytochrome MtoD and its physiological redox partners, to explore the EET processes of S. lithotrophicus ES-1.

Wood inspired biobased nanocomposite films composed of xylans, lignosulfonates and cellulose nanofibers for active food packaging.

The growing concerns on environmental pollution and sustainability have raised the interest on the development of functional biobased materials for different applications, including food packaging, as an alternative to the fossil resources-based counterparts, currently available in the market. In this work, functional wood inspired biopolymeric nanocomposite films were prepared by solvent casting of suspensions containing commercial beechwood xylans, cellulose nanofibers (CNF) and lignosulfonates (magnesium or sodium), in a proportion of 2:5:3 wt%, respectively. All films presented good homogeneity, translucency, and thermal stability up to 153 °C. The incorporation of CNF into the xylan/lignosulfonates matrix provided good mechanical properties to the films (Young's modulus between 1.08 and 3.79 GPa and tensile strength between 12.75 and 14.02 MPa). The presence of lignosulfonates imparted the films with antioxidant capacity (DPPH radical scavenging activity from 71.6 to 82.4 %) and UV barrier properties (transmittance ≤19.1 % (200-400 nm)). Moreover, the films obtained are able to successfully delay the browning of packaged fruit stored over 7 days at 4 °C. Overall, the obtained results show the potential of using low-cost and eco-friendly resources for the development of sustainable active food packaging materials.

A noncanonical IRAK4-IRAK1 pathway counters DNA damage-induced apoptosis independently of TLR/IL-1R signaling.

Interleukin-1 receptor (IL-1R)-associated kinases (IRAKs) are core effectors of Toll-like receptors (TLRs) and IL-1R in innate immunity. Here, we found that IRAK4 and IRAK1 together inhibited DNA damage-induced cell death independently of TLR or IL-1R signaling. In human cancer cells, IRAK4 was activated downstream of ATR kinase in response to double-strand breaks (DSBs) induced by ionizing radiation (IR). Activated IRAK4 then formed a complex with and activated IRAK1. The formation of this complex required the E3 ubiquitin ligase Pellino1, acting structurally but not catalytically, and the activation of IRAK1 occurred independently of extracellular signaling, intracellular TLRs, and the TLR/IL-1R signaling adaptor MyD88. Activated IRAK1 translocated to the nucleus in a Pellino2-dependent manner. In the nucleus, IRAK1 bound to the PIDD1 subunit of the proapoptotic PIDDosome and interfered with platform assembly, thus supporting cell survival. This noncanonical IRAK signaling pathway was also activated in response to other DSB-inducing agents. The loss of IRAK4, of IRAK4 kinase activity, of either Pellino protein, or of the nuclear localization sequence in IRAK1 sensitized p53-mutant zebrafish to radiation. Thus, the findings may lead to strategies for overcoming tumor resistance to conventional cancer treatments.

A Non-Canonical IRAK Signaling Pathway Triggered by DNA Damage.

Interleukin-1 receptor (IL-1R)-associated kinases (IRAKs) are core effectors of Toll-like receptor (TLR) and IL-1R signaling, with no reported roles outside of innate immunity. We find that vertebrate cells exposed to ionizing radiation (IR) sequentially activate IRAK4 and IRAK1 through a phosphorylation cascade mirroring that induced by TLR/IL-1R, resulting in a potent anti-apoptotic response. However, IR-induced IRAK1 activation does not require the receptors or the IRAK4/1 adaptor protein MyD88, and instead of remaining in the cytoplasm, the activated kinase is immediately transported to the nucleus via a conserved nuclear localization signal. We identify: double-strand DNA breaks (DSBs) as the biologic trigger for this pathway; the E3 ubiquitin ligase Pellino1 as the scaffold enabling IRAK4/1 activation in place of TLR/IL-1R-MyD88; and the pro-apoptotic PIDDosome (PIDD1-RAIDD-caspase-2) as a critical downstream target in the nucleus. The data delineate a non-canonical IRAK signaling pathway derived from, or ancestral to, TLR signaling. This DSB detection pathway, which is also activated by genotoxic chemotherapies, provides multiple actionable targets for overcoming tumor resistance to mainstay cancer treatments.

Relaxation-based NMR assignment: Spotlights on ligand binding sites in human CISD3.

CISD3 is a mitochondrial protein belonging to the NEET proteins family, bearing two [Fe2S2] clusters coordinated by CDGSH domains. At variance with the other proteins of the NEET family, very little is known about its structure-function relationships. NMR is the only technique to obtain information at the atomic level in solution on the residues involved in intermolecular interactions; however, in paramagnetic proteins this is limited by the broadening of signals of residues around the paramagnetic center. Tailored experiments can revive signals of the cluster surrounding; however, signals identification without specific residue assignment remains useless. Here, we show how paramagnetic relaxation can drive the signal assignment of residues in the proximity of the paramagnetic center(s). This allowed us to identify the potential key players of the biological function of the CISD3 protein.

Minimally invasive treatment of an amebic empyema secondary to the transdiaphragmatic rupture of a liver abscess: a case report.

Liver abscesses are a common complication in patients with amebiasis. Rarely, these will rupture across the diaphragm causing life-threatening empyemas. Evidence justifies performing surgical debridement or decortication for their treatment, given the better overall performance in comparison to open surgeries. However, no current guideline specifies which is the best approach. This report presents the case of a 39-year-old male with clinical, radiographical and microbiological evidence of an amebic empyema secondary to an amebic liver abscess, who received treatment by video-assisted thoracoscopy. The case description highlights the surgical technique, findings and operative outcomes that could be taken into consideration by other physicians to timely manage similar cases. The latter is especially relevant in underdeveloped and developing countries, where the burden of amebiasis appears to be greater. To the best of the authors' knowledge, this is the first description of a transdiaphragmatic amebic infection treated in a minimally invasive fashion.

Performance of the artificial urinary sphincter implantation in men with urinary incontinence: Results from a contemporary long-term real-world nationwide analysis.

PURPOSE: The artificial urinary sphincter (AUS) is one of the most effective surgical treatments for male urinary incontinence regardless of its severity. Current knowledge comes from high-volume centers, but little is known about the performance of this surgery from community practices. This study aims to report contemporary AUS performance in a nationwide observational study in Colombia. METHODS: Male patients who underwent AUS surgery with AMS 800™ between 2000 and 2020 in more than 17 centers and four cities were identified. Pre, intra, and postoperative characteristics were evaluated, mainly addressing patient reported outcomes measurements in the postoperative period. Retrospective and prospective data collection and descriptive analysis were completed. Kaplan-Meier analysis was used to determine AUS survival rate. RESULTS: Out of an initial 667 cases, a total of 215 patients met inclusion and exclusion criteria and were included. Mean age was 67 ± 9.4 years, and mean follow-up was 6.0 ± 4.4 years with maximum range of 14 years. The etiology of urinary incontinence was prostate cancer surgery in 141 (81%) of the cases. The rest of the cases were related to benign prostatic disease or spinal cord injury. It is noteworthy that out of 115 patients, only 59 (51.3%) reported previous formal pelvic floor rehabilitation. Subjective severity of urinary incontinence determined by a visual analog scale showed a decrease in 4.5 points after sphincter implantation. Sphincter removal was required in 50 (23.2%) cases. The main reasons for implant removal were urethral erosion and infection. The sphincter survival rate at 2, 5, 8, 10, and 14 years was 76%, 70%, 60%, 57%, and 17%, respectively. Of the subjects at the last follow-up with the device still in place, 80.7% defined their urinary condition as "does not cause or causes minor discomfort," and 99% would recommend the device to a friend or relative in the same condition. CONCLUSIONS: This series from a community-based practice shows the lack of adherence to clinical practice guidelines and the lack of standardized data collection. In contrast, this study provides real-world data on explantation and revision rates, allows physicians to inform patients and to have clear metrics for a shared decision-making process before the procedure.

Smurf2 inhibition enhances chemotherapy and radiation sensitivity in non-small-cell lung cancer.

Lung cancer has been the most common cancer worldwide for several decades. The outcomes of patients with locally advanced lung cancer remain dismal, and only a minority of patients survive more than 5 years. However, tumor therapeutic resistance mechanisms are poorly studied. Identification of therapeutic resistance pathways in lung cancer in order to increase the sensitivity of lung tumor cells to therapeutic agents is a crucial but challenging need. To identify novel genes that modulate the response to platinum-based therapy, we performed a genome-wide high-throughput ribonucleic acid interference (RNAi) screen via transfection of human lung cancer (PC9) cells with a viral short hairpin RNA (shRNA) library. We further validated a potential target via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic survival assays on PC9 and A549 lung tumor cells transfected with small interfering RNAs (siRNAs) to successfully downregulate protein expression and then treated with increasing doses of cisplatin or X-ray radiation. We determined protein expression by immunohistochemistry (IHC) after chemoradiotherapy and analyzed gene expression-based survival outcomes in two cohorts of human non-small-cell lung cancer (NSCLC) patients. The screen identified several targets involved in epithelial-to-mesenchymal transition (EMT), including Smurf1, Smurf2, YAP1, and CEBPZ, and glycolytic pathway proteins, including PFKFB3. Furthermore, we found that the small molecule proteasome inhibitor bortezomib significantly downregulated Smurf2 in lung cancer cells. The addition of bortezomib in combination with cisplatin and radiation therapy in PC9 and A549 cells led to an increase in deoxyribonucleic acid (DNA) double-strand breaks with increased numbers of γ-H2AX-positive cells and upregulation of apoptosis. Finally, we found that Smurf2 protein expression was upregulated in situ after treatment with cisplatin and radiation therapy in a relevant cohort of patients with stage III NSCLC. Additionally, Smurf2 gene expression was the strongest predictor of survival in patients with squamous NSCLC after chemotherapy or chemoradiotherapy. We successfully identified and validated Smurf2 as both a common modulator of resistance and an actionable target in lung cancer. These results suggest the urgent need to investigate clinical Smurf2 inhibition via bortezomib in combination with cisplatin and radiation for patients with locally advanced NSCLC.

A microRNA Cluster Controls Fat Cell Differentiation and Adipose Tissue Expansion By Regulating SNCG.

The H19X-encoded miR-424(322)/503 cluster regulates multiple cellular functions. Here, it is reported for the first time that it is also a critical linchpin of fat mass expansion. Deletion of this miRNA cluster in mice results in obesity, while increasing the pool of early adipocyte progenitors and hypertrophied adipocytes. Complementary loss and gain of function experiments and RNA sequencing demonstrate that miR-424(322)/503 regulates a conserved genetic program involved in the differentiation and commitment of white adipocytes. Mechanistically, it is demonstrated that miR-424(322)/503 targets γ-Synuclein (SNCG), a factor that mediates this program rearrangement by controlling metabolic functions in fat cells, allowing adipocyte differentiation and adipose tissue enlargement. Accordingly, diminished miR-424(322) in mice and obese humans co-segregate with increased SNCG in fat and peripheral blood as mutually exclusive features of obesity, being normalized upon weight loss. The data unveil a previously unknown regulatory mechanism of fat mass expansion tightly controlled by the miR-424(322)/503 through SNCG.

The miR-424(322)/503 gene cluster regulates pro- versus anti-inflammatory skin DC subset differentiation by modulating TGF-ß signaling.

Transforming growth factor ß (TGF-ß) family ligands are key regulators of dendritic cell (DC) differentiation and activation. Epidermal Langerhans cells (LCs) require TGF-ß family signaling for their differentiation, and canonical TGF-ß1 signaling secures a non-activated LC state. LCs reportedly control skin inflammation and are replenished from peripheral blood monocytes, which also give rise to pro-inflammatory monocyte-derived DCs (moDCs). By studying mechanisms in inflammation, we previously screened LCs versus moDCs for differentially expressed microRNAs (miRNAs). This revealed that miR-424/503 is the most strongly inversely regulated (moDCs > LCs). We here demonstrate that miR-424/503 is induced during moDC differentiation and promotes moDC differentiation in human and mouse. Inversely, forced repression of miR-424 during moDC differentiation facilitates TGF-ß1-dependent LC differentiation. Mechanistically, miR-424/503 deficiency in monocyte/DC precursors leads to the induction of TGF-ß1 response genes critical for LC differentiation. Therefore, the miR-424/503 gene cluster plays a decisive role in anti-inflammatory LC versus pro-inflammatory moDC differentiation from monocytes.

Polycomb complexes redundantly maintain epidermal stem cell identity during development.

Polycomb repressive complex 1 (PRC1) and PRC2 are critical epigenetic developmental regulators. PRC1 and PRC2 largely overlap in their genomic binding and cooperate to establish repressive chromatin domains demarcated by H2AK119ub and H3K27me3. However, the functional contribution of each complex to gene repression has been a subject of debate, and understanding of its physiological significance requires further studies. Here, using the developing murine epidermis as a paradigm, we uncovered a previously unappreciated functional redundancy between Polycomb complexes. Coablation of PRC1 and PRC2 in embryonic epidermal progenitors resulted in severe defects in epidermal stratification, a phenotype not observed in the single PRC1-null or PRC2-null epidermis. Molecular dissection indicated a loss of epidermal identity that was coupled to a strong derepression of nonlineage transcription factors, otherwise repressed by either PRC1 or PRC2 in the absence of its counterpart. Ectopic expression of subsets of PRC1/2-repressed nonepidermal transcription factors in wild-type epidermal stem cells was sufficient to suppress epidermal identity genes, highlighting the importance of functional redundancy between PRC1 and PRC2. Altogether, our studies show how PRC1 and PRC2 function as two independent counterparts, thereby providing a repressive safety net that protects and preserves lineage identity.

A model to predict SARS-CoV-2 infection based on the first three-month surveillance data in Brazil.

OBJECTIVE: COVID-19 diagnosis is a critical problem, mainly due to the lack or delay in the test results. We aimed to obtain a model to predict SARS-CoV-2 infection in suspected patients reported to the Brazilian surveillance system. METHODS: We analysed suspected patients reported to the National Surveillance System that corresponded to the following case definition: patients with respiratory symptoms and fever, who travelled to regions with local or community transmission or who had close contact with a suspected or confirmed case. Based on variables routinely collected, we obtained a multiple model using logistic regression. The area under the receiver operating characteristic curve (AUC) and accuracy indicators were used for validation. RESULTS: We described 1468 COVID-19 cases (confirmed by RT-PCR) and 4271 patients with other illnesses. With a data subset including 80% of patients from Sao Paulo (SP) and Rio Janeiro (RJ), we obtained a function which reached an AUC of 95.54% (95% CI: 94.41-96.67%) for the diagnosis of COVID-19 and accuracy of 90.1% (sensitivity 87.62% and specificity 92.02%). In a validation dataset including the other 20% of patients from SP and RJ, this model exhibited an AUC of 95.01% (92.51-97.5%) and accuracy of 89.47% (sensitivity 87.32% and specificity 91.36%). CONCLUSION: We obtained a model suitable for the clinical diagnosis of COVID-19 based on routinely collected surveillance data. Applications of this tool include early identification for specific treatment and isolation, rational use of laboratory tests, and input for modelling epidemiological trends.

OBJECTIF: Le diagnostic du COVID-19 est un problème critique, principalement dû au manque ou au retard dans les résultats du test. Nous visions à obtenir un modèle pour prédire l'infection par le SRAS-CoV-2 chez les patients suspects signalés au système de surveillance brésilien. MÉTHODES: Nous avons analysé les patients suspects signalés au Système National de Surveillance qui correspondaient à la définition de cas suivante: patients présentant des symptômes respiratoires et de la fièvre, qui se sont rendus dans des régions à transmission locale ou communautaire ou qui ont eu des contacts étroits avec un cas suspect ou confirmé. Sur la base de variables collectées en routine, nous avons obtenu un modèle multiple en utilisant la régression logistique. L'aire sous la courbe caractéristique de fonctionnement du récepteur (AUC) et les indicateurs de précision ont été utilisés pour la validation. RÉSULTATS: Nous avons décrit 1.468 cas de COVID-19 (confirmés par RT-PCR) et 4.271 patients atteints d'autres maladies. Avec un sous-ensemble de données comprenant 80% de patients de Sao Paulo (SP) et de Rio de Janeiro (RJ), nous avons obtenu une fonction qui atteignait une AUC de 95,54% (IC95%: 94,41% - 96,67%) pour le diagnostic de COVID- 19 et une précision de 90,1% (sensibilité 87,62% et spécificité 92,02%). Dans un ensemble de données de validation incluant les 20% restants de patients de SP et de RJ, ce modèle présentait une AUC de 95,01% (92,51% - 97,5%) et une précision de 89,47% (sensibilité 87,32% et spécificité 91,36%). CONCLUSION: Nous avons obtenu un modèle adapté au diagnostic clinique du COVID-19 sur la base des données de surveillance collectées en routine. Les applications de cet outil comprennent l'identification précoce pour un traitement et un isolement spécifiques, l'utilisation rationnelle des tests de laboratoire et des données pour modéliser les tendances épidémiologiques.

Transformed bone marrow cells generate neoplasms of distinct histogenesis. a murine model of cancer transplantation.

The last several years have witnessed renewed interest regarding the contribution of cancer stem cells in tumorigenesis and neoplastic heterogeneity. It has been reported that patients who undergo bone marrow transplantation are more prone to develop a malignancy during their life time; usually hematological tumors, but solid neoplasms may also develop, which in certain instances are donor-derived. It has also been well documented that multipotent bone marrow derived cells can migrate to diverse organs, differentiating into various histological lineages. The present study reports an experimental syngeneic transplantation model, using fluorescently tagged bone marrow cells from p53 null male mice into female wild-type counterparts. We found that transplanted non-neoplastic mutant bone marrow cells can generate tumors of distinct histogenesis, including thymic lymphomas, sarcomas, and carcinomas after carcinogen induction, providing evidence that multipotent cancer-prone stem cells can reside in the bone marrow and are transplantable.

An IRAK1-PIN1 signalling axis drives intrinsic tumour resistance to radiation therapy.

Drug-based strategies to overcome tumour resistance to radiotherapy (R-RT) remain limited by the single-agent toxicity of traditional radiosensitizers (for example, platinums) and a lack of targeted alternatives. In a screen for compounds that restore radiosensitivity in p53 mutant zebrafish while tolerated in non-irradiated wild-type animals, we identified the benzimidazole anthelmintic oxfendazole. Surprisingly, oxfendazole acts via the inhibition of IRAK1, a kinase thus far implicated in interleukin-1 receptor (IL-1R) and Toll-like receptor (TLR) immune responses. IRAK1 drives R-RT in a pathway involving IRAK4 and TRAF6 but not the IL-1R/TLR-IRAK adaptor MyD88. Rather than stimulating nuclear factor-κB, radiation-activated IRAK1 prevented apoptosis mediated by the PIDDosome complex (comprising PIDD, RAIDD and caspase-2). Countering this pathway with IRAK1 inhibitors suppressed R-RT in tumour models derived from cancers in which TP53 mutations predict R-RT. Moreover, IRAK1 inhibitors synergized with inhibitors of PIN1, a prolyl isomerase essential for IRAK1 activation in response to pathogens and, as shown here, in response to ionizing radiation. These data identify an IRAK1 radiation-response pathway as a rational chemoradiation therapy target.

Structure and reactivity of a siderophore-interacting protein from the marine bacterium Shewanella reveals unanticipated functional versatility.

Siderophores make iron accessible under iron-limited conditions and play a crucial role in the survival of microorganisms. Because of their remarkable metal-scavenging properties and ease in crossing cellular envelopes, siderophores hold great potential in biotechnological applications, raising the need for a deeper knowledge of the molecular mechanisms underpinning the siderophore pathway. Here, we report the structural and functional characterization of a siderophore-interacting protein from the marine bacterium Shewanella frigidimarina NCIBM400 (SfSIP). SfSIP is a flavin-containing ferric-siderophore reductase with FAD- and NAD(P)H-binding domains that have high homology with other characterized SIPs. However, we found here that it mechanistically departs from what has been described for this family of proteins. Unlike other FAD-containing SIPs, SfSIP did not discriminate between NADH and NADPH. Furthermore, SfSIP required the presence of the Fe2+-scavenger, ferrozine, to use NAD(P)H to drive the reduction of Shewanella-produced hydroxamate ferric-siderophores. Additionally, this is the first SIP reported that also uses a ferredoxin as electron donor, and in contrast to NAD(P)H, its utilization did not require the mediation of ferrozine, and electron transfer occurred at fast rates. Finally, FAD oxidation was thermodynamically coupled to deprotonation at physiological pH values, enhancing the solubility of ferrous iron. On the basis of these results and the location of the SfSIP gene downstream of a sequence for putative binding of aerobic respiration control protein A (ArcA), we propose that SfSIP contributes an additional layer of regulation that maintains cellular iron homeostasis according to environmental cues of oxygen availability and cellular iron demand.

High intensity resistance training causes muscle damage and increases biomarkers of acute kidney injury in healthy individuals.

PURPOSE: High-intensity interval resistance training (HIIRT) is an increasingly popular exercise program that provides positive results with short sessions. This study aimed to evaluate whether an HIIRT session causes muscle and kidney damage. METHODS: Fifty-eight healthy volunteers (median age 24 years, 50% women) participated in this study and performed a HIIRT session. The Borg CR10 scale for pain (CR10P) and blood and urine samples were collected before (baseline) and 2 and 24 hours after the HIIRT session. Blood samples were analyzed for serum creatinine (SCr), creatine kinase (CK) and myoglobin. Urine samples were assessed for creatinine, neutrophil gelatinase-associated lipocalin, interleukin 18, calbindin, microalbuminuria, trefoil factor-3 and ß-2 microglobulin. RESULTS: CR10P had a significant increase at 2 and 24 hours post-workout, and CK increased significantly at 2 hours and increased further at 24 hours. Myoglobin increased significantly at 2 hours and remained elevated at 24 hours. SCr increased modestly but significantly at 24 hours only in men. Three men met the KDIGO diagnostic criteria for acute kidney injury. The urinary kidney injury biomarkers increased significantly at 2 hours and returned to the baseline values 24 hours after HIIRT. CONCLUSIONS: A single HIIRT session caused early and significant elevations in CK, myoglobin, SCr, microalbuminuria and urinary biomarkers indicative of kidney tubular injury, suggesting the occurrence of muscle and kidney damage.

Physical activity prevents blood pressure increases in individuals under treatment for knee osteoarthritis.

The aim of this study was to assess the role of physical activity in blood pressure (BP) in individuals with knee osteoarthritis. We compared 136 participants under treatment for primary knee osteoarthritis (age=67.6±9.6 years) allocated to the sedentary-sedentary, active-sedentary, sedentary-active, and active-active groups depending on their levels of daily physical activity before and after follow-up. Their BP, BMI, and endurance performance (6-min walking test) were compared during 12 months of follow-up. The sedentary-sedentary group had increased systolic BP (11±3 mmHg), and the active-sedentary group had increased systolic (12±4 mmHg) and diastolic BP (5±1 mmHg) during follow-up. By contrast, the sedentary-active group maintained systolic BP and showed reduced diastolic BP (5±2 mmHg), and the active-active group maintained both systolic and diastolic BP. Positive effects on BP were accompanied by improvements in endurance performance and BMI in the sedentary-active group (endurance performance=8.5±2.7%; BMI=9.3±3.6%) and the active-active group (endurance performance=2.9±0.9%; BMI=3.8±2.0%), which did not occur in the sedentary-sedentary and active-sedentary groups. These results suggest a positive role of high levels of daily living physical activity in the prevention/management of hypertension in individuals with knee osteoarthritis.

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.

Bayesian Network to Infer Drug-Induced Apoptosis Circuits from Connectivity Map Data.

The Connectivity Map (CMAP) project profiled human cancer cell lines exposed to a library of anticancer compounds with the goal of connecting cancer with underlying genes and potential treatments. As most targeted anticancer therapeutics aim to induce tumor-selective apoptosis, it is critical to understand the specific cell death pathways triggered by drugs. This can help to better understand the mechanism of how cancer cells respond to chemical stimulations and improve the treatment of human tumors. In this study, using Connectivity MAP microarray-based gene expression data, we applied a Bayesian network modeling approach and identified apoptosis as a major drug-induced cellular pathway. We focused on 13 apoptotic genes that showed significant differential expression across all drug-perturbed samples to reconstruct the apoptosis network. In our predicted subnetwork, 9 out of 15 high-confidence interactions were validated in literature, and our inferred network captured two major cell death pathways by identifying BCL2L11 and PMAIP1 as key interacting players for the intrinsic apoptosis pathway, and TAXBP1 and TNFAIP3 for the extrinsic apoptosis pathway. Our inferred apoptosis network also suggested the role of BCL2L11 and TNFAIP3 as "gateway" genes in the drug-induced intrinsic and extrinsic apoptosis pathways.

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.