Genome-wide Analysis Identifies Nuclear Factor 1C as a Novel Transcription Factor and Potential Therapeutic Target in Small Cell Lung Cancer.

BACKGROUND: Recent insights regarding mechanisms mediating stemness, heterogeneity, and metastatic potential of lung cancers have yet to be fully translated to effective regimens for the treatment of these malignancies. This study sought to identify novel targets for lung cancer therapy. METHODS: Transcriptomes and DNA methylomes of 14 SCLC and 10 NSCLC lines were compared to normal human small airway epithelial cells (SAEC) and induced pluripotent stem cell (iPSC) clones derived from SAEC. SCLC lines, lung iPSC (Lu-iPSC), and SAEC were further evaluated by DNase I hypersensitivity (DHS-seq). Changes in chromatin accessibility and depths of transcription factor (TF) footprints were quantified using Bivariate analysis of Genomic Footprint. Standard techniques were used to examine growth and tumorigenencity as well as changes in transcriptomes and glucose metabolism of SCLC cells following Nuclear Factor 1C (NFIC) knockdown, and to examine NFIC expression in SCLC cells following exposure to BET inhibitors. RESULTS: Significant commonality of transcriptomes and DNA methylomes was observed between Lu-iPSC and SCLC; however, this analysis was uninformative regarding pathways unique to lung cancer. Linking results of DNase-seq to RNA-seq enabled identification of networks not previously associated with SCLC. When combined with footprint depth, NFIC, a transcription factor not previously associated with SCLC, had the highest score of occupancy at open chromatin sites. Knockdown of NFIC impaired glucose metabolism, decreased stemness, and inhibited growth of SCLC cells in-vitro and in-vivo. ChIP-seq analysis identified numerous sites occupied by Bromodomain-containing protein 4 (BRD4) in the NFIC promoter region. Knock-down of BRD4 or treatment with Bromodomain and extra-terminal domain (BET) inhibitors (BETi) markedly reduced NFIC expression in SCLC cells and SCLC PDX models. Approximately 8% of genes downregulated by BETi treatment were repressed by NFIC knockdown in SCLC, while 34% of genes repressed following NFIC knockdown were also downregulated in SCLC cells following BETi treatment. CONCLUSIONS: NFIC is a key TF and possible mediator of transcriptional regulation by BET family proteins in SCLC. Our findings highlight the potential of genome-wide chromatin accessibility analysis for elucidating mechanisms of pulmonary carcinogenesis and identifying novel targets for lung cancer therapy.

A Unique Gene Signature Predicting Recurrence Free Survival in Stage IA Lung Adenocarcinoma.

Objective: Resected stage IA lung adenocarcinoma (LUAD) has a reported 5-year recurrence free survival (RFS) of 63-81%. A unique gene signature stratifying patients with early stage LUAD as high or low-risk of recurrence would be valuable. Methods: GEO datasets combining European and North American LUAD patients (n=684) were filtered for stage IA (n=105) to develop a robust signature for recurrence (RFSscore). Univariate Cox proportional hazard regression model was used to assess associations of gene expression with RFS and OS. Leveraging a bootstrap approach of these identified upregulated genes allowed construction of a model which was evaluated by Area Under the Received Operating Characteristics. The optimal signature has RFSscore calculated via a linear combination of expression of selected genes weighted by the corresponding Cox regression derived coefficients. Log-rank analysis calculated RFS and OS. Results were validated using the LUAD TCGA transcriptomic NGS based dataset. Results: Rigorous bioinformatic analysis identified a signature of 4 genes: KNSTRN, PAFAH1B3, MIF, CHEK1. Kaplan-Meier analysis of stage IA LUAD with this signature resulted in 5-year RFS for low-risk of 90% compared to 53% for high-risk (HR 6.55, 95%CI 2.65-16.18, p-value <0.001), confirming the robustness of the gene signature with its clinical significance. Validation of the signature using TCGA dataset resulted in an AUC of 0.797 and 5-year RFS for low and high-risk stage IA patients being 91% and 67%, respectively (HR 3.44, 95%CI 1.16-10.23, p-value=0.044). Conclusions: This 4 gene signature stratifies European and North American patients with pathologically confirmed stage IA LUAD into low and high-risk groups for OS and more importantly RFS.

Cigarette Smoke Enhances the Malignant Phenotype of Esophageal Adenocarcinoma Cells by Disrupting a Repressive Regulatory Interaction Between miR-145 and LOXL2.

Although linked to esophageal carcinogenesis, the mechanisms by which cigarette smoke mediates initiation and progression of esophageal adenocarcinomas (EAC) have not been fully elucidated. In this study, immortalized esophageal epithelial cells and EAC cells (EACCs) were cultured with or without cigarette smoke condensate (CSC) under relevant exposure conditions. Endogenous levels of microRNA (miR)-145 and lysyl-likeoxidase 2 (LOXL2) were inversely correlated in EAC lines/tumors compared with that in immortalized cells/normal mucosa. The CSC repressed miR-145 and upregulated LOXL2 in immortalized esophageal epithelial cells and EACCs. Knockdown or constitutive overexpression of miR-145 activated or depleted LOXL2, respectively, which enhanced or reduced proliferation, invasion, and tumorigenicity of EACC, respectively. LOXL2 was identified as a novel target of miR-145 as well as a negative regulator of this miR in EAC lines/Barrett's epithelia. Mechanistically, CSC induced recruitment of SP1 to the LOXL2 promoter; LOXL2 upregulation coincided with LOXL2 enrichment and concomitant reduction of H3K4me3 levels within the promoter of miR143HG (host gene for miR-145). Mithramycin downregulated LOXL2 and restored miR-145 expression in EACC and abrogated LOXL2-mediated repression of miR-145 by CSC. These findings implicate cigarette smoke in the pathogenesis of EAC and demonstrate that oncogenic miR-145-LOXL2 axis dysregulation is potentially druggable for the treatment and possible prevention of these malignancies.

Socioeconomic-Related Inequalities in COVID-19 Vulnerability in South Africa.

Individuals' vulnerability to the risk of COVID-19 infection varies due to their health, socioeconomic, and living circumstances, which also affect the effectiveness of implementing non-pharmacological interventions (NPIs). In this study, we analysed socioeconomic-related inequalities in COVID-19 vulnerability using data from the nationally representative South African General Household Survey 2019. We developed a COVID-19 vulnerability index, which includes health and social risk factors for COVID-19 exposure and susceptibility. The concentration curve and concentration index were used to measure socioeconomic-related inequalities in COVID-19 vulnerability. Recentred influence function regression was then utilised to decompose factors that explain the socioeconomic-related inequalities in COVID-19 vulnerability. The concentration index estimates were all negative and highly significant (p < 0.01), indicating that vulnerability to COVID-19 was more concentrated among the poor. According to the decomposition analysis, higher income and education significantly (p < 0.01) positively impacted lowering socioeconomic-related COVID-19 vulnerability. Living in an urban region, being Black, and old all had significant (p < 0.01) positive impacts on increasing socioeconomic-related COVID-19 vulnerability. Our findings contribute to a better understanding of socially defined COVID-19-vulnerable populations in South Africa and the implications for future pandemic preparedness plans.

Poly(rC) binding protein 1 benefits coxsackievirus B3 infection via suppressing the translation of p62/SQSTM1.

Coxsackievirus B3 (CVB3) is a positive single-strand RNA virus causing myocarditis, pancreatitis and meningitis. During CVB3 infection, various host cellular components, including proteins and non-coding RNAs, interact with the virus and affect viral infection. Poly(rC) binding protein 1 (PCBP1) is a multifunctional RNA binding protein regulating transcription, translation and mRNA stability of a variety of genes. In this study, we observed a significant reduction of PCBP1 protein during CVB3 infection. By bioinformatic prediction and luciferase-assay verification, we confirmed that the expression of PCBP1 was directly inhibited by miR-21, a microRNA upregulated during CVB3 infection. Furthermore, we found that overexpression of PCBP1 promoted CVB3 infection and knocking down of PCBP1 inhibited it. In the subsequent mechanism study, our results revealed that PCBP1 blocked the translation of p62/SQSTM1 (sequestosome 1), an autophagy-receptor protein suppressing CVB3 replication, by interacting with the cis-element in the 5' untranslational region (5' UTR) of p62/SQSTM1. In summary, our studies have identified PCBP1 as a beneficial factor for CVB3 infection. These findings may deepen the understanding of host-virus interactions and provide a potential target for intervention of CVB3 infection.

Kernel Flow: a high channel count scalable time-domain functional near-infrared spectroscopy system.

SIGNIFICANCE: Time-domain functional near-infrared spectroscopy (TD-fNIRS) has been considered as the gold standard of noninvasive optical brain imaging devices. However, due to the high cost, complexity, and large form factor, it has not been as widely adopted as continuous wave NIRS systems. AIM: Kernel Flow is a TD-fNIRS system that has been designed to break through these limitations by maintaining the performance of a research grade TD-fNIRS system while integrating all of the components into a small modular device. APPROACH: The Kernel Flow modules are built around miniaturized laser drivers, custom integrated circuits, and specialized detectors. The modules can be assembled into a system with dense channel coverage over the entire head. RESULTS: We show performance similar to benchtop systems with our miniaturized device as characterized by standardized tissue and optical phantom protocols for TD-fNIRS and human neuroscience results. CONCLUSIONS: The miniaturized design of the Kernel Flow system allows for broader applications of TD-fNIRS.

Hookah Smoke Mediates Cancer-Associated Epigenomic and Transcriptomic Signatures in Human Respiratory Epithelial Cells.

INTRODUCTION: Although communal smoking of hookah by means of water pipes is perceived to be a safe alternative to cigarette smoking, the effects of hookah smoke in respiratory epithelia have not been well characterized. This study evaluated epigenomic and transcriptomic effects of hookah smoke relative to cigarette smoke in human respiratory epithelial cells. METHODS: Primary normal human small airway epithelial cells from three donors and cdk4 and hTERT-immortalized small airway epithelial cells and human bronchial epithelial cells were cultured for 5 days in normal media with or without cigarette smoke condensates (CSCs) or water pipe condensates (WPCs). Cell count, immunoblot, RNA sequencing, quantitative real-time reverse-transcriptase polymerase chain reaction, methylation-specific polymerase chain reaction, and quantitative chromatin immunoprecipitation techniques were used to compare effects of hookah and cigarette smoke on cell proliferation, global histone marks, gene expression, and promoter-related chromatin structure. RESULTS: CSC and WPC decreased global H4K16ac and H4K20me3 histone marks and mediated distinct and overlapping cancer-associated transcriptome signatures and pathway modulations that were cell line dependent and stratified across lung cancer cells in a histology-specific manner. Epiregulin encoding a master regulator of EGFR signaling that is overexpressed in lung cancers was up-regulated, whereas FILIP1L and ABI3BP encoding mediators of senescence that are repressed in lung cancers were down-regulated by CSC and WPC. Induction of epiregulin and repression of FILIP1L and ABI3BP by these condensates coincided with unique epigenetic alterations within the respective promoters. CONCLUSIONS: These findings support translational studies to ascertain if hookah-mediated epigenomic and transcriptomic alterations in cultured respiratory epithelia are detectable and clinically relevant in hookah smokers.

Randomized phase II trial of a first-in-human cancer cell lysate vaccine in patients with thoracic malignancies.

BACKGROUND: Although most malignancies express cancer-testis antigens (CTA), immune responses to these proteins are limited in thoracic oncology patients. This trial was undertaken to examine if a cancer cell lysate vaccine could induce immunity to CTA, and to ascertain if metronomic cyclophosphamide and celecoxib enhances vaccine-induced immune responses. METHODS: Eleven patients with primary thoracic malignancies and 10 patients with extrathoracic neoplasms metastatic to the chest rendered NED by conventional therapies were randomized to receive H1299 lung cancer cell lysates (10 mg protein/vaccine) with Iscomatrix™ adjuvant via deep intradermal injection q 4 weeks ×6 with or without daily oral metronomic cyclophosphamide/celecoxib. The primary endpoint was serologic response to purified CTA assessed 1 month after the 6th vaccination. Secondary endpoints included assessment of the effects of cyclophosphamide and celecoxib on frequency and magnitude of vaccine-induced immune responses to CTA. Exploratory endpoints included evaluation of the effects of the vaccine regimens on peripheral immune subsets. Standard of care imaging studies were obtained at baseline and 1 month after the 3rd and 6th vaccinations. RESULTS: All patients exhibited local and systemic inflammatory responses lasting 72-96 hours following vaccinations. There were no dose limiting treatment related toxicities. Fourteen patients (67%) completed all six vaccinations. Eight of 14 patients (57%) exhibited serologic responses to NY-ESO-1. One patient developed antibodies to GAGE7; several patients exhibited reactivity to XAGE and MAGE-C2. Vaccine therapy decreased the percent of Tregs (P=0.0068), PD-1 expression on Tregs (P=0.0027), PD-L1 expression on CD14+ monocytes (P=0.0089), PD-L1 expression on classical monocytes (P=0.016), and PD-L1 expression on intermediate monocytes (P=0.0031). Cyclophosphamide/celecoxib did not appear to increase immune responses or enhance vaccine-induced alterations in peripheral immune subsets. CONCLUSIONS: H1299 lysate vaccines with Iscomatrix™ induce immune responses to CTA and modulate peripheral immune subsets in a manner that may enhance antitumor immunity in patients with thoracic malignancies.

A critical role of AREG for bleomycin-induced skin fibrosis.

We report our discovery of an important player in the development of skin fibrosis, a hallmark of scleroderma. Scleroderma is a fibrotic disease, affecting 70,000 to 150,000 Americans. Fibrosis is a pathological wound healing process that produces an excessive extracellular matrix to interfere with normal organ function. Fibrosis contributes to nearly half of human mortality. Scleroderma has heterogeneous phenotypes, unpredictable outcomes, no validated biomarkers, and no effective treatment. Thus, strategies to slow down scleroderma progression represent an urgent medical need. While a pathological wound healing process like fibrosis leaves scars and weakens organ function, oral mucosa wound healing is a scarless process. After re-analyses of gene expression datasets from oral mucosa wound healing and skin fibrosis, we discovered that several pathways constitutively activated in skin fibrosis are transiently induced during oral mucosa wound healing process, particularly the amphiregulin (Areg) gene. Areg expression is upregulated ~ 10 folds 24hrs after oral mucosa wound but reduced to the basal level 3 days later. During bleomycin-induced skin fibrosis, a commonly used mouse model for skin fibrosis, Areg is up-regulated throughout the fibrogenesis and is associated with elevated cell proliferation in the dermis. To demonstrate the role of Areg for skin fibrosis, we used mice with Areg knockout, and found that Areg deficiency essentially prevents bleomycin-induced skin fibrosis. We further determined that bleomycin-induced cell proliferation in the dermis was not observed in the Areg null mice. Furthermore, we found that inhibiting MEK, a downstream signaling effector of Areg, by selumetinib also effectively blocked bleomycin-based skin fibrosis model. Based on these results, we concluded that the Areg-EGFR-MEK signaling axis is critical for skin fibrosis development. Blocking this signaling axis may be effective in treating scleroderma.

Housing, sanitation and living conditions affecting SARS-CoV-2 prevention interventions in 54 African countries.

The feasibility of non-pharmacological public health interventions (NPIs) such as physical distancing or isolation at home to prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission in low-resource countries is unknown. Household survey data from 54 African countries were used to investigate the feasibility of SARS-CoV-2 NPIs in low-resource settings. Across the 54 countries, approximately 718 million people lived in households with ⩾6 individuals at home (median percentage of at-risk households 56% (95% confidence interval (CI), 51% to 60%)). Approximately 283 million people lived in households where ⩾3 people slept in a single room (median percentage of at-risk households 15% (95% CI, 13% to 19%)). An estimated 890 million Africans lack on-site water (71% (95% CI, 62% to 80%)), while 700 million people lacked in-home soap/washing facilities (56% (95% CI, 42% to 73%)). The median percentage of people without a refrigerator in the home was 79% (95% CI, 67% to 88%), while 45% (95% CI, 39% to 52%) shared toilet facilities with other households. Individuals in low-resource settings have substantial obstacles to implementing NPIs for mitigating SARS-CoV-2 transmission. These populations urgently need to be prioritised for coronavirus disease 2019 vaccination to prevent disease and to contain the global pandemic.

UHRF1 Is a Novel Druggable Epigenetic Target in Malignant Pleural Mesothelioma.

INTRODUCTION: Ubiquitin-like with plant homeodomain and ring finger domains 1 (UHRF1) encodes a master regulator of DNA methylation that has emerged as an epigenetic driver in human cancers. To date, no studies have evaluated UHRF1 expression in malignant pleural mesothelioma (MPM). This study was undertaken to explore the therapeutic potential of targeting UHRF1 in MPM. METHODS: Microarray, real-time quantitative reverse transcription-polymerase chain reaction, immunoblot, and immunohistochemistry techniques were used to evaluate UHRF1 expression in normal mesothelial cells (NMCs) cultured with or without asbestos, MPM lines, normal pleura, and primary MPM specimens. The impact of UHRF1 expression on MPM patient survival was evaluated using two independent databases. RNA-sequencing, proliferation, invasion, and colony formation assays, and murine xenograft experiments were performed to evaluate gene expression and growth of MPM cells after biochemical or pharmacologic inhibition of UHRF1 expression. RESULTS: UHRF1 expression was significantly higher in MPM lines and specimens relative to NMC and normal pleura. Asbestos induced UHRF1 expression in NMC. The overexpression of UHRF1 was associated with decreased overall survival in patients with MPM. UHRF1 knockdown reversed genomewide DNA hypomethylation, and inhibited proliferation, invasion, and clonogenicity of MPM cells, and growth of MPM xenografts. These effects were phenocopied by the repurposed chemotherapeutic agent, mithramycin. Biochemical or pharmacologic up-regulation of p53 significantly reduced UHRF1 expression in MPM cells. RNA-sequencing experiments exhibited the pleiotropic effects of UHRF1 down-regulation and identified novel, clinically relevant biomarkers of UHRF1 expression in MPM. CONCLUSIONS: UHRF1 is an epigenetic driver in MPM. These findings support the efforts to target UHRF1 expression or activity for mesothelioma therapy.

2020 American College of Rheumatology Guideline for the Management of Gout.

OBJECTIVE: To provide guidance for the management of gout, including indications for and optimal use of urate-lowering therapy (ULT), treatment of gout flares, and lifestyle and other medication recommendations. METHODS: Fifty-seven population, intervention, comparator, and outcomes questions were developed, followed by a systematic literature review, including network meta-analyses with ratings of the available evidence according to the Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology, and patient input. A group consensus process was used to compose the final recommendations and grade their strength as strong or conditional. RESULTS: Forty-two recommendations (including 16 strong recommendations) were generated. Strong recommendations included initiation of ULT for all patients with tophaceous gout, radiographic damage due to gout, or frequent gout flares; allopurinol as the preferred first-line ULT, including for those with moderate-to-severe chronic kidney disease (CKD; stage >3); using a low starting dose of allopurinol (≤100 mg/day, and lower in CKD) or febuxostat (<40 mg/day); and a treat-to-target management strategy with ULT dose titration guided by serial serum urate (SU) measurements, with an SU target of <6 mg/dl. When initiating ULT, concomitant antiinflammatory prophylaxis therapy for a duration of at least 3-6 months was strongly recommended. For management of gout flares, colchicine, nonsteroidal antiinflammatory drugs, or glucocorticoids (oral, intraarticular, or intramuscular) were strongly recommended. CONCLUSION: Using GRADE methodology and informed by a consensus process based on evidence from the current literature and patient preferences, this guideline provides direction for clinicians and patients making decisions on the management of gout.

2020 American College of Rheumatology Guideline for the Management of Gout.

OBJECTIVE: To provide guidance for the management of gout, including indications for and optimal use of urate-lowering therapy (ULT), treatment of gout flares, and lifestyle and other medication recommendations. METHODS: Fifty-seven population, intervention, comparator, and outcomes questions were developed, followed by a systematic literature review, including network meta-analyses with ratings of the available evidence according to the Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology, and patient input. A group consensus process was used to compose the final recommendations and grade their strength as strong or conditional. RESULTS: Forty-two recommendations (including 16 strong recommendations) were generated. Strong recommendations included initiation of ULT for all patients with tophaceous gout, radiographic damage due to gout, or frequent gout flares; allopurinol as the preferred first-line ULT, including for those with moderate-to-severe chronic kidney disease (CKD; stage >3); using a low starting dose of allopurinol (≤100 mg/day, and lower in CKD) or febuxostat (<40 mg/day); and a treat-to-target management strategy with ULT dose titration guided by serial serum urate (SU) measurements, with an SU target of <6 mg/dl. When initiating ULT, concomitant antiinflammatory prophylaxis therapy for a duration of at least 3-6 months was strongly recommended. For management of gout flares, colchicine, nonsteroidal antiinflammatory drugs, or glucocorticoids (oral, intraarticular, or intramuscular) were strongly recommended. CONCLUSION: Using GRADE methodology and informed by a consensus process based on evidence from the current literature and patient preferences, this guideline provides direction for clinicians and patients making decisions on the management of gout.

Expression Profile and Function Analysis of Long Non-coding RNAs in the Infection of Coxsackievirus B3.

The roles of lncRNAs in the infection of enteroviruses have been barely demonstrated. In this study, we used coxsackievirus B3 (CVB3), a typical enterovirus, as a model to investigate the expression profiles and functional roles of lncRNAs in enterovirus infection. We profiled lncRNAs and mRNA expression in CVB3-infected HeLa cells by lncRNA-mRNA integrated microarrays. As a result, 700 differentially expressed lncRNAs (431 up-regulated and 269 down-regulated) and 665 differentially expressed mRNAs (299 up-regulated and 366 down-regulated) were identified in CVB3 infection. Then we performed lncRNA-mRNA integrated pathway analysis to identify potential functional impacts of the differentially expressed mRNAs, in which lncRNA-mRNA correlation network was built. According to lncRNA-mRNA correlation, we found that XLOC-001188, an lncRNA down-regulated in CVB3 infection, was negatively correlated with NFAT5 mRNA, an anti-CVB3 gene reported previously. This interaction was supported by qPCR detection following siRNA-mediated knockdown of XLOC-001188, which showed an increase of NFAT5 mRNA and a reduction of CVB3 genomic RNA. In addition, we observed that four most significantly altered lncRNAs, SNHG11, RP11-145F16.2, RP11-1023L17.1 and RP11-1021N1.2 share several common correlated genes critical for CVB3 infection, such as BRE and IRF2BP1. In all, our studies reveal the alteration of lncRNA expression in CVB3 infection and its potential influence on CVB3 replication, providing useful information for future studies of enterovirus infection.

Severe Hepatotoxicity of Mithramycin Therapy Caused by Altered Expression of Hepatocellular Bile Transporters.

Mithramycin demonstrates preclinical anticancer activity, but its therapeutic dose is limited by the development of hepatotoxicity that remains poorly characterized. A pharmacogenomics characterization of mithramycin-induced transaminitis revealed that hepatotoxicity is associated with germline variants in genes involved in bile disposition: ABCB4 (multidrug resistance 3) rs2302387 and ABCB11 [bile salt export pump (BSEP)] rs4668115 reduce transporter expression (P < 0.05) and were associated with ≥grade 3 transaminitis developing 24 hours after the third infusion of mithramycin (25 mcg/kg, 6 hours/infusion, every day ×7, every 28 days; P < 0.0040). A similar relationship was observed in a pediatric cohort. We therefore undertook to characterize the mechanism of mithramycin-induced acute transaminitis. As mithramycin affects cellular response to bile acid treatment by altering the expression of multiple bile transporters (e.g., ABCB4, ABCB11, sodium/taurocholate cotransporting polypeptide, organic solute transporter α/ß) in several cell lines [Huh7, HepaRG, HepaRG BSEP (-/-)] and primary human hepatocytes, we hypothesized that mithramycin inhibited bile-mediated activation of the farnesoid X receptor (FXR). FXR was downregulated in all hepatocyte cell lines and primary human hepatocytes (P < 0.0001), and mithramycin inhibited chenodeoxycholic acid- and GW4046-induced FXR-galactose-induced gene 4 luciferase reporter activity (P < 0.001). Mithramycin promoted glycochenodeoxycholic acid-induced cytotoxicity in ABCB11 (-/-) cells and increased the overall intracellular concentration of bile acids in primary human hepatocytes grown in sandwich culture (P < 0.01). Mithramycin is a FXR expression and FXR transactivation inhibitor that inhibits bile flow and potentiates bile-induced cellular toxicity, particularly in cells with low ABCB11 function. These results suggest that mithramycin causes hepatotoxicity through derangement of bile acid disposition; results also suggest that pharmacogenomic markers may be useful to identify patients who may tolerate higher mithramycin doses. SIGNIFICANCE STATEMENT: The present study characterizes a novel mechanism of drug-induced hepatotoxicity in which mithramycin not only alters farnesoid X receptor (FXR) and small heterodimer partner gene expression but also inhibits bile acid binding to FXR, resulting in deregulation of cellular bile homeostasis. Two novel single-nucleotide polymorphisms in bile flow transporters are associated with mithramycin-induced liver function test elevations, and the present results are the rationale for a genotype-directed clinical trial using mithramycin in patients with thoracic malignancies.

ASXL3 Is a Novel Pluripotency Factor in Human Respiratory Epithelial Cells and a Potential Therapeutic Target in Small Cell Lung Cancer.

In this study, we generated induced pluripotent stem cells (iPSC) from normal human small airway epithelial cells (SAEC) to investigate epigenetic mechanisms of stemness and pluripotency in lung cancers. We documented key hallmarks of reprogramming in lung iPSCs (Lu-iPSC) that coincided with modulation of more than 15,000 genes relative to parental SAECs. Of particular novelty, we identified the PRC2-associated protein, ASXL3, which was markedly upregulated in Lu-iPSCs and small cell lung cancer (SCLC) lines and clinical specimens. ASXL3 overexpression correlated with increased genomic copy number in SCLC lines. ASXL3 silencing inhibited proliferation, clonogenicity, and teratoma formation by Lu-iPSCs, and diminished clonogenicity and malignant growth of SCLC cells in vivo Collectively, our studies validate the utility of the Lu-iPSC model for elucidating epigenetic mechanisms contributing to pulmonary carcinogenesis and highlight ASXL3 as a novel candidate target for SCLC therapy. Cancer Res; 77(22); 6267-81. ©2017 AACR.

Precision-scored parental report questions and HL-scaled tympanometry as informative measures of hearing in otitis media 1: Large-sample evidence on determinants and complementarity to pure-tone audiometry.

INTRODUCTION: In otitis media with effusion (OME), hearing loss is a core sign/symptom and basis of concern, with absolute pure-tone threshold sensitivity (in dB HL) by air-conduction providing the default measure of hearing. However several fundamental problems limiting the value of HL measures in otitis media are insufficiently appreciated. To appraise the joint value and implications of multiple hearing measures towards more comprehensive hearing assessment in OM, we examine in two related articles the interrelations and common or diverging determinants of three measures, two of them objective: binaural HL, and ACET (the published quasi-continuous scaling of binaural tympanometry to HL). The third measure is partly subjective: parentally reported hearing difficulties (RHD-4); this is the precision-scored total of the 4 items selected for the OM8-30 general purpose questionnaire for parents in OM. METHODS: The Eurotitis-2 study (Total N=2886) internationally standardises OM8-30 and its OMQ-14 short form. The clinical and parent-response variables acquired cover many issues in diagnosis, symptomatology and impact of OM. Data acquisition was built upon routine clinic practice, enabling us also to document some properties of that practice, such as patterns of missing HL data. To address possible confounding or loss of representativeness from this, we investigated the implications of substituting tympanometry-based ACET for missing HL to give an HL/ACET hybrid. ACET is the mapping of categorical tympanometry to continuous HL. We simulated degrees of artificial missingness of HL up to 35% on the 1430 complete-data cases, using random deletion, with 1000-version bootstrapping. Correlations of this HL/ACET hybrid with pure (100%) HL then documented the degree of correlation retained under dilution of HL by an admixture of ACET; we also documented distribution shapes. For RHD-4, we then probed the determining influences on severity of score as an auditory disability measure, both background ones (from centre, age, sex, socio-economic status, length of history, diagnosis and season) and the two underlying objective hearing measures (HL, ACET). We ran these multiple regressions (GLMs), for representativeness and generality, both on 1430 complete-data cases (i.e. all 3 hearing variables present) and also on supplemented samples according to data required only for particular analyses (N increased by +56% to +68%). A further method of sample supplementation (by up to +96%) used the HL/ACET hybrid. RESULTS: Sex made negligible difference in any analysis. The particular collaborating centre, age, season and diagnosis collectively influenced presence/absence of HL data very strongly. (Area under ROC 0.944). Socio-economic status did not influence HL presence; surprisingly, nor did RHD, ACET or length of history, after control for centre, age, diagnosis and season. Of the inter-correlations between hearing measures, only the one between ACET and RHD was influenced (slightly reduced) by the inclusion of cases without HL data. In the simulated substitutions, Pearson correlation of hybrid HL/ACET with true HL remained above 0.90 for substitution by ACET of up to 30% rate of artificially 'missing' HL. Centre differences were adequately summarised by simple absolute additive differences in mean local case severity. In the determinant models for RHD on the 1430 complete-data cases, HL and the set of background determinants collectively explained broadly similar proportions of RHD's variability, totalling 36.8% explained. On the larger maximum case samples, slightly less absolute variability was explicable than on complete-case data, but relative magnitudes of contribution from individual determinants, both background and hearing measures, remained similar. The expected mean differences in RHD between diagnoses (RAOM, OME, and combined) were found, but the patterns of background and objective measure influences determining RHD did not differ significantly between the diagnoses. CONCLUSIONS: (1) In the Eurotitis-2 database, descriptive differences in various background demographic and clinical measures between cases on whom HL data were obtained versus not, were only of material magnitude for length of history and reported hearing difficulties. Such descriptive differences are not necessarily bases of confounding, so using our framework of 6 background adjuster variables, (particular collaborating centre, age, season, diagnosis, socioeconomic status and length of history) we isolated the determinants of HL data presence. The first four listed strongly predicted HL data presence/absence so are sufficient to control analyses well for any bias or confounding by HL data presence. (2) Diagnoses as OME and combined (OME+RAOM) had higher probability of HL data being present relative to RAOM, indicating that HL acquisition is chiefly seen as confirming and quantifying hearing loss in (suspect) OME, not as ruling it out (e.g. in suspected RAOM). Given this, also using RHD and or ACET as pre-triage to efficiently target capacity and/or reduce costs and opportunity costs of acquiring HL would be rational, but there was no evidence of such precise use of initial hearing-related information to decide on HL acquisition. (3) The full six background variables explained comparable variance in Reported Hearing Difficulties (RHD) to what was explained by ACET, but not quite as much as by HL. Achieving a high percentage explained (32-37% from good models) required both classes of determinant to be entered as predictors. The pattern of background determining influences for RHD was largely stable, with or without objective measures as additional predictors, and on maximum or complete-data cases. Length of history strongly determines RHD for a given concurrent HL. (4) Accepting ACET as substitute where HL was missing in OM cases gave a sample-size enhancement of 17% in Eurotitis-2, with negligible difference in the pattern of determinants. This hybrid measure can be recommended as reasonable next-best when moderate percentages of HL data are missing. (5) The stable pattern of prediction of RHD suggests that our six background determinants provide a very promising low-cost yet comprehensive framework for determination. It hence offers pluripotent statistical adjustment against confounding, applicable to RAOM, OME and combined diagnoses in any analysis using this database. Claims that it thereby offers a sufficient framework for full European standardisation of all the scores from the OM8-30 questionnaire measures await parallel demonstrations for symptom areas other than RHD. As 25% of the variance in RHD severity can be explained by the six adjusters in our framework, none of the six variables should be omitted from acquisition and analytic use in future OM research.

Mithramycin Depletes Specificity Protein 1 and Activates p53 to Mediate Senescence and Apoptosis of Malignant Pleural Mesothelioma Cells.

PURPOSE: Specificity protein 1 (SP1) is an oncogenic transcription factor overexpressed in various human malignancies. This study sought to examine SP1 expression in malignant pleural mesotheliomas (MPM) and ascertain the potential efficacy of targeting SP1 in these neoplasms. EXPERIMENTAL DESIGN: qRT-PCR, immunoblotting, and immunohistochemical techniques were used to evaluate SP1 expression in cultured MPM cells and MPM specimens and normal mesothelial cells/pleura. MTS, chemotaxis, soft agar, ß-galactosidase, and Apo-BrdUrd techniques were used to assess proliferation, migration, clonogenicity, senescence, and apoptosis in MPM cells following SP1 knockdown, p53 overexpression, or mithramycin treatment. Murine subcutaneous and intraperitoneal xenograft models were used to examine effects of mithramycin on MPM growth in vivo. Microarray, qRT-PCR, immunoblotting, and chromatin immunoprecipitation techniques were used to examine gene expression profiles mediated by mithramycin and combined SP1 knockdown/p53 overexpression and correlate these changes with SP1 and p53 levels within target gene promoters. RESULTS: MPM cells and tumors exhibited higher SP1 mRNA and protein levels relative to control cells/tissues. SP1 knockdown significantly inhibited proliferation, migration, and clonogenicity of MPM cells. Mithramycin depleted SP1 and activated p53, dramatically inhibiting proliferation and clonogenicity of MPM cells. Intraperitoneal mithramycin significantly inhibited growth of subcutaneous MPM xenografts and completely eradicated mesothelioma carcinomatosis in 75% of mice. Mithramycin modulated genes mediating oncogene signaling, cell-cycle regulation, senescence, and apoptosis in vitro and in vivo. The growth-inhibitory effects of mithramycin in MPM cells were recapitulated by combined SP1 knockdown/p53 overexpression. CONCLUSIONS: These findings provide preclinical rationale for phase II evaluation of mithramycin in patients with mesothelioma.

Pulmonary Metastases Exhibit Epigenetic Clonality: Implications for Precision Cancer Therapy.

BACKGROUND: Development of effective cancer therapies may be limited by intratumoral heterogeneity, which facilitates outgrowth and organ-specific dissemination of treatment resistant clones. At present, limited information is available regarding epigenetic landscapes of pulmonary metastases. This study was undertaken to characterize epigenetic signatures of pulmonary metastases and to identify potential therapeutic targets. METHODS: RNA and DNA were extracted from 65 pulmonary metastases resected from 12 patients (5 with sarcoma, 7 with adrenocortical carcinoma). Quantitative reverse transcription polymerase chain reaction techniques were used to evaluate expression levels of cancer-testis (CT) genes (NY-ESO-1, MAGE-A3, MAGE-A9, MAGE-A12, GAGE1, CT-45, SSX-1, and SSX-2), tumor suppressor (TS) genes (p16 and RASSF1A), and genes encoding epigenetic modifiers (DNMT1, DNMT3A, DNMT3B, EZH2, EED, and SUZ12), aberrantly expressed in human malignant diseases. Pyrosequencing techniques were used to quantitate DNA methylation levels in LINE1, NBL2, and D4Z4 repetitive sequences and promoter methylation status of differentially regulated genes. Results of these analyses were compared with a standardized panel of normal lung tissues. RESULTS: Pulmonary metastases exhibited histologically related and patient-specific global DNA demethylation. Significant interpatient heterogeneity of gene expression was observed even among patients with similar tumor histologic features. Epigenetic signatures appeared consistent among metastases from the same patient, irrespective of the time of resection (synchronous/metachronous) or the anatomic location. EZH2, EED, and SUZ12 (core components of Polycomb repressive complex-2 [PRC-2]) were upregulated in the majority of metastases. CONCLUSIONS: Pulmonary metastases exhibit patient-specific epigenetic clonality, which may be exploited for precision therapies targeting aberrant CT or TS gene expression. PRC-2 may be a shared target for epigenetic therapy of pulmonary metastases.