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Purpose This study aims to develop a multi-gene assay predictive of the clinical benefits of chemotherapy in non-small cell lung cancer (NSCLC) patients, and substantiate their protein expression as potential therapeutic targets. Patients and methods The mRNA expression of 160 genes identified from microarray was analyzed in qRT-PCR assays of independent 337 snap-frozen NSCLC tumors to develop a predictive signature. A clinical trial JBR.10 was included in the validation. Hazard ratio was used to select genes, and decision-trees were used to construct the predictive model. Protein expression was quantified with AQUA in 500 FFPE NSCLC samples. Results A 7-gene signature was identified from training cohort (n = 83) with accurate patient stratification (P = 0.0043) and was validated in independent patient cohorts (n = 248, P, Highlights • This paper develops a RT-PCR based 7-gene assay to predict the clinical benefits of chemotherapy for precision oncology • This work discovers a prognostic protein biomarker ZNF71 using AQUA technique as a potential companion test and therapeutic target • This research identifies prognostic mRNA and protein biomarker CD27 with implications in immunotherapy The results from this paper will help clinicians to select NSCLC patients who might benefit from chemotherapy. The identified prognostic protein biomarkers might also be new therapeutic targets in chemotherapy and immunotherapy.

BACKGROUND: Plethora of efforts fails to yield a single drug to reverse the pathogenesis of Parkinson's disease (PD) and related α-synucleopathies. METHODS: Using chemical biology, we identified a small molecule inhibitor of c-abl kinase, PD180970 that could potentially clear the toxic protein aggregates. Genetic, molecular, cell biological and immunological assays were performed to understand the mechanism of action. In vivo preclinical disease model of PD was used to assess its neuroprotection efficacy. FINDINGS: In this report, we show the ability of a small molecule inhibitor of tyrosine kinases, PD180970, to induce autophagy (cell lines and mice midbrain) in an mTOR-independent manner and ameliorate the α-synuclein mediated toxicity. PD180970 also exerts anti-neuroinflammatory potential by inhibiting the release of proinflammatory cytokines such as IL-6 (interleukin-6) and MCP-1 (monocyte chemoattractant protein-1) through reduction of TLR-4 (toll like receptor-4) mediated NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation. In vivo studies show that PD180970 is neuroprotective by degrading the toxic protein oligomers through induction of autophagy and subsiding the microglial activation. INTERPRETATION: These protective mechanisms ensure the negation of Parkinson's disease related motor impairments. FUND: This work was supported by Wellcome Trust/DBT India Alliance Intermediate Fellowship (500159-Z-09-Z), DST-SERB grant (EMR/2015/001946), DBT (BT/INF/22/SP27679/2018) and JNCASR intramural funds to RM, and SERB, DST (SR/SO/HS/0121/2012) to PAA, and DST-SERB (SB/YS/LS-215/2013) to JPC and BIRAC funding to ETA C-CAMP. ispartof: EBIOMEDICINE vol:50 pages:260-273 ispartof: location:Netherlands status: published

Background: BM32, a grass pollen allergy vaccine containing four recombinant fusion proteins consisting of hepatitis B-derived PreS and hypoallergenic peptides from the major timothy grass pollen allergens adsorbed on aluminium hydroxide has been shown to be safe and to improve clinical symptoms of grass pollen allergy upon allergen-specific immunotherapy (AIT). We have investigated the immune responses in patients from a two years double-blind, placebo-controlled AIT field trial with BM32. Methods: Blood samples from patients treated with BM32 (n = 27) or placebo (Aluminium hydroxide) (n = 13) were obtained to study the effects of vaccination and natural allergen exposure on allergen-specific antibody, T cell and cytokine responses. Allergen-specific IgE, IgG, IgG1 and IgG4 levels were determined by ImmunoCAP and ELISA, respectively. Allergen-specific lymphocyte proliferation by 3H thymidine incorporation and multiple cytokine responses with a human 17-plex cytokine assay were studied in cultured peripheral blood mononuclear cells (PBMCs). Findings: Two years AIT comprising two courses of 3 pre-seasonal injections of BM32 and a single booster after the first pollen season induced a continuously increasing (year 2 > year 1) allergen-specific IgG4 response without boosting allergen-specific IgE responses. Specific IgG4 responses were accompanied by low stimulation of allergen-specific PBMC responses. Increases of allergen-specific pro-inflammatory cytokine responses were absent. The rise of allergen-specific IgE induced by seasonal grass pollen exposure was partially blunted in BM32-treated patients. Interpretation: AIT with BM32 is characterised by the induction of a non-inflammatory, continuously increasing allergen-specific IgG4 response (year 2 > year1) which may explain that clinical efficacy was higher in year 2 than in year 1. The good safety profile of BM32 may be explained by lack of IgE reactivity and low stimulation of allergen-specific T cell and cytokine responses. Fundings: Grants F4605, F4613 and DK 1248-B13 of the Austrian Science Fund (FWF). Keywords: Allergy, Allergen, Allergen-specific immunotherapy (AIT), Recombinant allergy vaccine, BM32, IgE, IgG subclass, T cell response, Cytokine response

Background: Human cytomegalovirus (HCMV) causes a ubiquitous infection which can pose a significant threat for immunocompromised individuals, such as those undergoing solid organ transplant (SOT). Arguably, the most successful vaccine studied to date is the recombinant glycoprotein-B (gB) with MF59 adjuvant which, in 3 Phase II trials, demonstrated 43–50% efficacy in preventing HCMV acquisition in seronegative healthy women or adolescents and reduction in virological parameters after SOT. However, the mechanism of vaccine protection in seronegative recipients remains undefined. Methods: We evaluated samples from the cohort of seronegative SOT patients enroled in the Phase II glycoprotein-B/MF59 vaccine trial who received organs from seropositive donors. Samples after SOT (0–90 days) were tested by real-time quantitative PCR for HCMV DNA. Anti-gB antibody levels were measured by ELISA. Neutralization was measured as a decrease in infectivity for fibroblast cell cultures revealed by expression of immediate-early antigens. Findings: Serological analyses revealed a more rapid increase in the humoral response against gB post transplant in vaccine recipients than in those randomised to receive placebo. Importantly, a number of patient sera displayed HCMV neutralising responses – neutralisation which was abrogated by pre-absorbing the sera with recombinant gB. Interpretation: We hypothesise that the vaccine primed the immune system of seronegative recipients which, when further challenged with virus at time of transplant, allowed the host to mount rapid immunological humoral responses even under conditions of T cell immune suppression during transplantation. Keywords: Cytomegalovirus, Vaccination, Antibody responses, Prime-boost

Background: Ovarian clear cell carcinoma (OCCC) is a histological subtype of epithelial ovarian cancer (EOC) with distinct pathological, biological, and molecular features. OCCCs are more resistant to conventional treatment regimen of EOC and have the worst stage-adjusted prognosis amongst EOC subtypes. As the OCCC incidence rate in Asian populations has significantly increased in recent decades, it is critical to elucidate its molecular features that could lead to OCCC-tailored therapeutic strategies. Methods: Gene expression profiles of 222 OCCC were analyzed by hierarchical clustering and statistical analyses. Findings: We identified two OCCC gene expression subtypes: EpiCC—epithelial-like, which is associated with early-stage disease, with a relatively higher rate of gene mutations in the SWI/SNF complex; and MesCC—mesenchymal-like, associated with late-stage and higher enrichment of immune-related pathway activity. Genetic, copy number and transcriptomic analyses showed that both EpiCC and MesCC carried OCCC-associated aberrations. The EpiCC/MesCC classification was reproducible in validation cohorts and OCCC cell lines. MesCC tumors had a poorer progression-free survival (PFS) than EpiCC tumors (HR: 3·0, p = 0·0006). Functional assays in cell lines showed that the MesCC subtype was more proliferative and more anoikis-resistant than the EpiCC. By applying the EpiCC/MesCC classification to the TCGA renal clear cell carcinoma cohort, our results indicated interoperability of the subtyping scheme, and revealed preferential drug response of MesCC to bevacizumab. Interpretation: The EpiCC/MesCC classification shows promise for prognostic and therapeutic stratification in OCCC patients and warrants further investigation in the context of OCCC gene expression subtype-tailored treatment strategies. Keywords: Ovarian cancer, Clear cell, Microarray gene expression, Molecular subtype

Background Recently, the distinction between left- and right-sided colon cancer (LCC and RCC) has been brought into focus. RCC is associated with an inferior overall survival and progression-free survival. We aimed to perform a detailed analysis of the diversity of extracellular vesicles (EV) between LCC and RCC using quantitative proteomics and to identify for new diagnostic and prognostic biomarkers. Methods We isolated EVs from patients with LCC, RCC and healthy volunteers, and treated colorectal cancer cell line with serum-derived EVs. We then performed a quantitative proteomics analysis of the serum-derived EVs and cell line treated with EVs. Proteomic data are available via ProteomeXchange with the identifiers PXD012283 and PXD012304. In addition, we assessed the performance of EV SPARC and LRG1 as diagnosis and prognosis biomarkers in colon cancer. Findings The expression profile of the serum EV proteome in patients with RCC was different from that of patients with LCC. Serum-derived EVs in RCC promoted cellular mobility more significantly than EVs derived from LCC. EV SPARC and LRG1 expression levels demonstrated area under the receiver-operating characteristic curve values of 0.95 and 0.93 for discriminating patients with colon cancer from healthy controls. Moreover, the expression levels of SPARC and LRG1 correlated with tumour sidedness and were predictive of tumour recurrence. Interpretation We identified differences in EV protein profiles between LCC and RCC. Serum-derived EVs of RCC may promote metastasis via upregulation of extracellular matrix (ECM)-related proteins, especially SPARC and LRG1, which may serve as diagnosis and prognosis biomarkers in colon cancer.

Background: HIV drug resistance (HIVDR) testing can assist clinicians in selecting treatments. However, high complexity and cost of genotyping assays limit routine testing in settings where HIVDR prevalence has reached high levels. Methods: The oligonucleotide ligation assay (OLA)-Simple kit was developed for detection of HIVDR against first-line non-nucleoside/nucleoside reverse transcriptase inhibitors and validated on 672 codons (168 specimens) from subtypes A, B, C, D, and AE. The kit uses dry reagents to facilitate assay setup, lateral flow devices for visual HIVDR detections, and in-house software with an interface for guiding users and analyzing results. Findings: HIVDR analysis of specimens by OLA-Simple compared to Sanger sequencing revealed 99.6 ± 0.3% specificity and 98.2 ± 0.9% sensitivity, and compared to high-sensitivity assays, 99.6 ± 0.6% specificity and 86.2 ± 2.5% sensitivity, with 2.6 ± 0.9% indeterminate results. OLA-Simple was performed more rapidly compared to Sanger sequencing (

Background: Dyslipidemia is commonly observed in various kidney diseases, renal specific secreted erythropoietin (EPO) may participate in this process. However, how this process is regulated has remained mostly elusive. Method: The dyslipidemia was evaluated in chronic kidney disease and ischemia kidney injury animal model. Primary cultured adipocytes was used to investigate the lipolytic effect of EPO. Lipidemia was evaluated in EPO treated animal. Blood samples from cardiac surgery-induced kidney injury were collected to assess correlationship between EPO and lipidemia. Findings: We found a decrease in secreted EPO, led to hypertriglyceridemia in chronic kidney disease (CKD) mice. In contrast, in ischemic kidney animal model, increased EPO triggered by hypoxia signaling activation, was accompanied by decreased triglyceride (TG) in plasma. Mechanistically, circulating EPO modulates JAK2-STAT5 signaling, which in turn enhances lipolysis in peripheral adipose tissue and contributes to dysregulated lipidemia in serum. Delivering of recombinant EPO into both normal and CKD mice suppressed plasma TG by accelerating lipolysis in adipose tissue. In a cohort of patients diagnosed with acute kidney injury after cardiopulmonary bypass surgery, the decreased TG and cholesterol negatively correlated with increased EPO in the serum. Interpretation: This study identifies that renal secreted EPO controls lipidemia in kidney disease, providing a new mechanism underlying dyslipidemia in chronic kidney disease. Circulating EPO stimulates lipolysis by targeting JAK2-STATA5 signaling in peripheral adipose tissue, providing new therapeutic target for dyslipidemia treatment. Funding Statement: This work was supported by grants from the National Natural Science Foundation of China (Nos. 81700640, 81970608). Declaration of Interests: The authors declare that there is no duality of interest associated with this manuscript. Ethics Approval Statement: All clinical study were approved by the Medical Ethics Committee of Nanfang Hospital (Guangzhou, China). The participants in the study provided written informed consent. All animal experiments were approved by the Institutional Animal Care Committee of Nanfang Hospital.

Background: Pediatric high-grade gliomas (pHGG) are the leading cause of cancer-related death during childhood. Due to their diffuse growth characteristics, chemoresistance and location behind the blood-brain barrier (BBB), the prognosis of pHGG has barely improved in the past decades. As such, there is a dire need for new therapies that circumvent those difficulties. Since aberrant expression of DNA damage-response associated Fanconi anemia proteins play a central role in the onset and therapy resistance of many cancers, we here investigated if FANCD2 depletion could sensitize pHGG to additional DNA damage. Methods: We determined the capacity of celastrol, a BBB-penetrable compound that degrades FANCD2, to sensitize glioma cells to the archetypical DNA-crosslinking agent carboplatin in vitro in seven patient-derived pHGG models. In addition, we tested this drug combination in vivo in a patient-derived orthotopic pHGG xenograft model. Underlying mechanisms to drug response were investigated using mRNA expression profiling, western blotting, immunofluorescence, FANCD2 knockdown and DNA fiber assays. Findings: FANCD2 is overexpressed in HGGs and depletion of FANCD2 by celastrol synergises with carboplatin to induce cytotoxicity. Combination therapy prolongs survival of pHGG-bearing mice over monotherapy and control groups in vivo (P

Background Plasmodium falciparum deficient for hrp2 and hrp3 genes are a threat to malaria management and elimination, since they escape widely used HRP2-based rapid diagnostic tests and treatment. Hrp2/hrp3 deletions are increasingly reported from all malaria endemic regions but are currently only identified by laborious methodologies. Methods We developed a novel hydrolysis probe-based, quantitative, real-time PCR (4plex qPCR) for detection and discrimination of P. falciparum infection (cytb) and hrp2 and hrp3 gene status, and to control assay validity (btub). A cross-sectional, diagnostic accuracy study was performed in Gabon for assay validation and deletion screening. Findings In parallel to identification of P. falciparum infection in samples down to 0.05 parasites/µl, the 4plex qPCR enabled specific and valid interrogation of the parasites´s hrp2 and hrp3 genes in one go - even in low parasitemic samples. The assay was precise and robust also when performed in a routine healthcare setting in Gabon. The risk of falsely identifying hrp2 or hrp3 deletion was reduced by 100-fold compared to conventional PCR. Evaluation against microscopy was performed on 200 blood samples collected in Gabon: sensitivity and specificity of 4plex qPCR (cytb) were 100% and 80%, respectively. Stringent testing revealed hrp2 deletion in 2 of 95 P. falciparum positive and validated samples. Interpretation The novel 4plex qPCR is sensitive, accurate and allows resource-efficient rapid screening. Monitoring and mapping of hrp2/hrp3 deletions is required to identify areas where control strategies may need to be adapted to ensure appropriate patient care and ultimately achieve malaria elimination. Funding BMBF (03VP00402).

Background: Mesoderm Posterior 1 (MESP1) belongs to the family of basic helix-loop-helix transcription factors. It is a master regulator of mesendoderm development, leading to formation of organs such as heart and lung. However, its role in adult pathophysiology remains unknown. Here, we report for the first time a previously-unknown association of MESP1 with non-small cell lung cancer (NSCLC). Methods: MESP1 mRNA and protein levels were measured in NSCLC-derived cells by qPCR and immunoblotting respectively. Colony formation assay, colorimetric cell proliferation assay and soft agar colony formation assays were used to assess the effects of MESP1 knockdown and overexpression in vitro. RNA-sequencing and chromatin immunoprecipitation (ChIP)-qPCR were used to determine direct target genes of MESP1. Subcutaneous injection of MESP1-depleted NSCLC cells in immuno-compromised mice was done to study the effects of MESP1 mediated tumor formation in vivo. Findings: We found that MESP1 expression correlates with poor prognosis in NSCLC patients, and is critical for proliferation and survival of NSCLC-derived cells, thus implicating MESP1 as a lung cancer oncogene. Ectopic MESP1 expression cooperates with loss of tumor suppressor ARF to transform murine fibroblasts. Xenografts from MESP1-depleted cells showed decreased tumor growth in vivo. Global transcriptome analysis revealed a MESP1 DNA-binding-dependent gene signature associated with various hallmarks of cancer, suggesting that transcription activity of MESP1 is most likely responsible for its oncogenic abilities. Interpretation: Our study demonstrates MESP1 as a previously-unknown lineage-survival oncogene in NSCLC which may serve as a potential prognostic marker and therapeutic target for lung cancer in the future. Keywords: Mesp1, ARF, Lung cancer, Lineage-survival oncogene

Background Some interstitial lung disease (ILD) patients develop a progressive fibrosing-ILD phenotype (PF-ILD), with similar persistent lung function decline suggesting common molecular pathways involved. Nintedanib, a tyrosine kinase inhibitor targeting the PDGF, FGF, VEGF and M-CSF pathways, has shown comparable efficacy in idiopathic pulmonary fibrosis (IPF) and systemic sclerosis-associated ILD (SSc-ILD). We hypothesize that Nintedanib targeted molecular pathways will be augmented to a similar degree across PF-ILD regardless of aetiology. Methods We collected explanted lung tissue at the time of lung transplantation from 130 PF-ILD patients (99 (76%) IPF, 14 (11%) SSc-ILD, 17 (13%) other PF-ILD), and wedge biopsies from 200 donor lungs and measured PDGF, FGF, VEGF and M-CSF concentrations by Luminex. Findings The concentrations of PDGF-AA, PDGF-BB, FGF-2, VEGF and M-CSF were significantly increased in PF-ILD lungs compared to donor lungs (PDGF-AA 93·0 pg/ml [±97·2] vs. 37·5 pg/ml [±35·4], p

Background: Recurrent implantation failure (RIF) remains a critical and challenging problem in assisted reproductive technology mainly because of impaired decidualization. Endocytic and transcytotic activity in the endometrium are crucial for impaired decidualization. The most representative endocytic genes is the C-terminal Eps15 homology domain-containing 1 (EHD1). Whether the EHD1-mediated endocytic function is responsible for embryo implantation during decidualization remains to be determined. Methods: Endometrial tissues were collected from RIF patients (n=27) and fertile controls (n=27) between July 2017 and December 2018. Meanwhile, endometrial biopsies were performed on tissues from 10 women with infertility secondary to tubal factor in the late proliferative and mid-secretory phases of the menstrual cycle. Primary HESCs isolated from eutopic endometrial tissues were used to investigate the role of EHD1 in decidualization. Findings: RNA-seq analysis demonstrated that EHD1 expression was significantly higher in the mid-secretory endometrium of the RIF group than in that of the fertile control group. During the menstrual cycle, the expression of EHD1 increased in the mid-proliferative phase and decreased periodically in the mid-secretory phase and decidual phase. Furthermore, EHD1 overexpression impaired decidualization by suppressing the expression of prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1) and the formation of the cytoskeleton. Mechanistically, Wingless-related MMTV integration site 4 (Wnt4) and its downregulated signaling showed significant changes following adenovirus-mediated overexpression of EHD1 in HESCs by RNA-seq analysis. The endocytosis pathway of the low-density-lipoprotein receptor-related proteins 5 and 6 (LRP5/6) co-receptor was decreased by EHD1 overexpression. Co-IP and confocal microscopy analysis showed that EHD1 bound to the Wnt4 protein, contributing to the decrease in Wnt4/β-catenin signaling during the decidualization of HESCs. Additionally, the Wnt4 agonist improved the impaired decidualization. Interpretation: Regulation of the EHD1-Wnt4 pathway may serve as a promising therapeutic strategy to improve endometrial receptivity in RIF women. Funding Statement: This work was supported by the National Key Research and Development Program of China (2018YFC1004401), the National Natural Science Foundation of China (81871165, 31872846, 81571402 and 31571189 ), a special grant for principal investigators from the Health Department of Jiangsu Province (ZDRCA2016070), and a special fund for the Jiangsu Province Social Development Project (NO.BE2018602). Declaration of Interests: All authors declare no conflicts of interest. Ethics Approval Statement: This study was carried out according to the Recommendations of Guidelines for Clinical Trials by the Ethics Committee of the Drum Tower Hospital. Informed consent was obtained from all participants before any study-related procedure was performed. This study was carried out according to the approval of Construction and Management of the Nanjing Multic-center Biobank Project (No. 2013-081-01).

Background: : Prostate cancer (PCa) is a leading cause of cancer-related death in males. Aberrant expression of long noncoding RNAs (lncRNAs) is frequently reported in human malignancies. This study was performed to explore the role of LSAMP-AS1 in epithelial-mesenchymal transition (EMT), proliferation, migration and invasion of PCa cells. Methods: : Initially, the differentially expressed lncRNAs in PCa were screened out by microarray analysis. The clinicopathological and prognostic significance of LSAMP-AS1 was evaluated. LSAMP-AS1 was over-expressed or silenced to investigate the roles in EMT, proliferation, migration and invasion of PCa cells. Moreover, the relationships between LSAMP-AS1 and miR-183–5p, as well as miR-183–5p and decorin (DCN) were characterized. The tumorigenicity of PCa cells was verified in nude mice. Results: : LSAMP-AS1 was poorly expressed in PCa tissues and cells. Low expression of LSAMP-AS1 was indicative of poor overall survival and disease-free survival, and related to Gleason score, TNM stage, and risk stratification. Over-expressed LSAMP-AS1 inhibited EMT, proliferation, migration and invasion of PCa cells, as well as tumor growth in nude mice. Meanwhile, over-expression of LSAMP-AS1 resulted in up-regulation of E-cadherin and down-regulation of Vimentin, N-cadherin, Ki67, PCNA, MMP-2, MMP-9, Ezrin and Fascin. Notably, LSAMP-AS1 competitively bound to miR-183–5p which directly targets DCN. It was confirmed that the inhibitory effect of LSAMP-AS1 on PCa cells was achieved by binding to miR-183–5p, thus promoting the expression of DCN. Conclusion: : LSAMP-AS1 up-regulates the DCN gene by competitively binding to miR-183–5p, thus inhibiting EMT, proliferation, migration and invasion of PCa cells. Keywords: Prostate cancer, LSAMP-AS1, MicroRNA-183–5p, Decorin, Epithelial-mesenchymal transition, Proliferation, Migration

Background: The understanding of the regulation of glucagon secretion by pancreatic islet α-cells remains elusive. We aimed to develop an in vitro model for investigating the function of human α-cells under direct influence of glucose and other potential regulators. Methods: Highly purified human α-cells from islets of deceased donors were re-aggregated in the presence or absence of β-cells in culture, evaluated for glucagon secretion under various treatment conditions, and compared to that of intact human islets and non-sorted islet cell aggregates. Findings: The pure human α-cell aggregates maintained proper glucagon secretion capability at low concentrations of glucose, but failed to respond to changes in ambient glucose concentration. Addition of purified β-cells, but not the secreted factors from β-cells at low or high concentrations of glucose, partly restored the responsiveness of α-cells to glucose with regulated glucagon secretion. The EphA stimulator ephrinA5-fc failed to mimic the inhibitory effect of β-cells on glucagon secretion. Glibenclamide inhibited glucagon secretion from islets and the α- and β-mixed cell-aggregates, but not from the α-cell-only aggregates, at 2.0 mM glucose. Interpretation: This study validated the use of isolated and then re-aggregated human islet cells for investigating α-cell function and paracrine regulation, and demonstrated the importance of cell-to-cell contact between α- and β-cells on glucagon secretion. Loss of proper β- and α-cell physical interaction in islets likely contributes to the dysregulated glucagon secretion in diabetic patients. Re-aggregated select combinations of human islet cells provide unique platforms for studying islet cell function and regulation. Keywords: Human alpha cells glucagon, Insulin inhibition, Somatostatin, Glibenclamide, Paracrine regulation, Cell-to-cell contact

Background: Metastasis of rhabdomyosarcoma (RMS) is the primary cause of tumour-related deaths. Previous studies have shown that overexpression of the guanine nucleotide exchange factor T (GEFT) is correlated with a poorer RMS prognosis, but the mechanism remains largely unexplored. Methods: We focused on determining the influence of the GEFT-Rho-GTPase signalling pathway and the epithelial–mesenchymal transition (EMT) or mesenchymal–epithelial transition (MET) on RMS progression and metastasis by using RMS cell lines, BALB/c nude mice and cells and molecular biology techniques. Findings: GEFT promotes RMS cell viability, migration, and invasion; GEFT also inhibits the apoptosis of RMS cells and accelerates the growth and lung metastasis of RMS by activating the Rac1/Cdc42 pathways. Interestingly, GEFT upregulates the expression levels of N-cadherin, Snail, Slug, Twist, Zeb1, and Zeb2 and reduces expression level of E-cadherin. Thus, GEFT influences the expression of markers for EMT and MET in RMS cells via the Rac1/Cdc42-PAK1 pathways. We also found that the level of GEFT gene promoter methylation in RMS is lower than that in normal striated muscle tissue. Significant differences were observed in the level of GEFT gene methylation in different histological subtypes of RMS. Interpretation: These findings suggest that GEFT accelerates the tumourigenicity and metastasis of RMS by activating Rac1/Cdc42-PAK signalling pathway-induced EMT; thus, it may serve as a novel therapeutic target. Fund: This work was supported by grants from the National Natural Science Foundation of China (81660441, 81460404, and 81160322) and Shihezi University Initiative Research Projects for Senior Fellows (RCZX201447). Funders had no role in the design of the study, data collection, data analysis, interpretation, or the writing of this report. Keywords: Rhabdomyosarcoma, GEFT, Rac1/Cdc42-PAK1 pathways, EMT, Methylation

Background: Despite toxic side effects and limited durable response, the current standard-of-care treatment for high grade serous ovarian cancer (HGSOC) remains platinum/taxane-based chemotherapy. Given that the overall prognosis has not improved drastically over the past several decades, there is a critical need to understand the underlying mechanisms that lead to tumour development and progression. Methods: We utilized an integrative proteogenomic analysis of HGSOC tumours applying a poor prognosis gene expression signature (PPS) as a conceptual framework to analyse orthogonal genomic and proteomic data from the TCGA (n = 488) and CPTAC (n = 169) studies. Genes identified through in silico analyses were assessed in vitro studies to demonstrate their impact on proliferation and cell cycle progression. Findings: These analyses identified DNA amplification and overexpression of the transcription factor ADNP (Activity Dependent Neuroprotector Homeobox) in poorly prognostic tumours. Validation studies confirmed the prognostic capacity of ADNP and suggested an oncogenic role for this protein given the association between ADNP expression and pro-proliferative signalling. In vitro studies confirmed ADNP as a novel and essential mediator of cell proliferation through dysregulation of cell cycle checkpoints. Interpretation: We identified ADNP as being amplified and overexpressed in poor prognosis HGSOC in silico analyses and demonstrated that ADNP is a novel and essential oncogene in HGSOC which mediates proliferation through dysregulation of cell cycle checkpoints in vitro. Funding: The National Cancer Institute of the National Institutes of Health, the V Foundation for Cancer Research and the New Jersey Commission for Cancer Research. Keywords: High-grade serous ovarian cancer, HGSOC, Proteogenomic, ADNP, Poor prognostic marker

Background: Endocrine-cerebro-osteodysplasia (ECO) syndrome is a genetic disorder associated with congenital defects of the endocrine, cerebral, and skeletal systems in humans. ECO syndrome is caused by mutations of the intestinal cell kinase (ICK) gene, which encodes a mitogen-activated protein (MAP) kinase-related kinase that plays a critical role in controlling the length of primary cilia. Lack of ICK function disrupts transduction of sonic hedgehog (SHH) signaling, which is important for development and homeostasis in humans and mice. Craniofacial structure abnormalities, such as cleft palate, are one of the most common defects observed in ECO syndrome patients, but the role of ICK in palatal development has not been studied. Methods: Using Ick-mutant mice, we investigated the mechanisms by which ICK function loss causes cleft palate and examined pharmacological rescue of the congenital defects. Findings: SHH signaling was compromised with abnormally elongated primary cilia in the developing palate of Ick-mutant mice. Cell proliferation was significantly decreased, resulting in failure of palatal outgrowth, although palatal adhesion and fusion occurred normally. We thus attempted to rescue the congenital palatal defects of Ick mutants by pharmacological activation of SHH signaling. Treatment of Ick-mutant mice with an agonist for Smoothened (SAG) rescued several congenital defects, including cleft palate. Interpretations: The recovery of congenital defects by pharmacological intervention in the mouse models for ECO syndrome highlights prenatal SHH signaling modulation as a potential therapeutic measure to overcome congenital defects of ciliopathies. Keywords: Cleft palate, Ciliopathy, Sonic hedgehog, Endocrine-cerebro-osteodysplasia (ECO) syndrome, Smoothened agonist (SAG)

Background: Chronic Hepatitis B (CHB) remains a major problem for global public health. Viral persistence and immune defects are the two major reasons for CHB, and it was hypothesized that based on a transient clearance of serum viral DNA and HBsAg “window stage”, active immunization against hepatitis B virus (HBV) might initiate effective host immune responses versus HBV to achieve functional cure of CHB. Methods: Two experimental mouse models that mice hydrodynamic injected HBV DNA or infected with recombinant AAV/HBV were used. The “sandwich” therapeutic effect by using a potent human anti-HBsAg neutralizing monoclonal antibody (G12) in combination with antiviral drug tenofovir disoproxil fumarate (TDF), followed by active immunization with HBsAg-HBsAb (mYIC) was evaluated. Findings: A single G12 injection rapidly cleared serum HBsAg in HDI-HBV carrier mice, with a synergistic effect in decreasing viral DNA load when TDF was given orally. When both serum viral DNA and HBsAg load became low or undetectable, mYIC was administered. A more effective clearance of viral DNA and HBsAg was observed and serum HBsAb was developed only in these “sandwich”-treated mice. Efficient intrahepatic anti-HBV immune responses were also observed in these mice, including the formation of aggregates of myeloid cells with CD8+ T cells and increased TNF-α, granzyme B production. Interpretation: The “sandwich” combination therapy not only efficiently decreased HBsAg and HBV DNA levels but also induced effective cellular and humoral immunity, which may result in functional cure of CHB. Keywords: Chronic hepatitis B, HBsAb, TDF, HBsAg-HBsAb immune complex, Liver CD8+ T cells

Background: Sunitinib is one of the multi-targeted tyrosine kinase inhibitors for the treatment of renal cell carcinoma (RCC) at present. However, its clinical efficacy is limited by chemoresistance of RCC. Our previous study found that eukaryotic translation initiation factor 3 subunit D (EIF3D) was an oncogene in the development and progression of RCC but little is known about whether EIF3D participated in sunitinib resistance of RCC. Methods: The expression of EIF3D in the tumor tissue specimen was detected by immunohistochemistry. The effect of EIF3D on sunitinib-resistance of RCC cells was evaluated by colony formation, IC50 proliferation and in vivo tumor growth assays. The interaction between EIF3D and glucose regulated protein 78 (GRP78) was assessed by Co-IP and Western blot assays. Finding: EIF3D expression was found higher in 786-OR and ACHN-R cells with acquired sunitinib resistance than that in 786-O and ACHN cells sunitinib to sensitive. The EIF3D level was also up-regulated in sunitinib-chemoresistant tumor tissues compared with chemosensitive tumor tissues. Functional study showed that EIF3D knockdown decreased cell viability with sunitinib treatment. Mechanistical study demonstrated that EIF3D interacted with GRP78 and enhanced protein stability through blocking the ubiquitin-mediated-proteasome degradation of GRP78. GRP78 overexpression induced sunitinib resistance of RCC cells by triggering the unfolded protein response, whereas GRP78 silencing inhibited cell viability. Forced expression of GRP78 eliminated the inhibitory effect of EIF3D silencing on cell growth in vitro and in vivo. Interpretation: our results indicate that EIF3D played an important role in sunitinib resistance of RCC cells, suggesting that it may prove to be a potential therapeutic target for RCC. Keywords: Renal cell carcinoma, EIF3D, GRP78, Sunitinib, Chemoresistance

Background: Mothers are the primary source of bacteria for newborns, but it is unclear whether mother-to-newborn transmission occurs prior to, during or after birth. Similarly, the effect of the delivery mode on neonatal microorganisms has been the focus of controversy. Methods: Healthy maternal and neonatal pairs that underwent vaginal birth and caesarean section were enrolled in this study. Meconium, placenta, membrane and amniotic fluid samples for newborns and vaginal, rectal and oral samples for mothers were collected. All samples were amplified and sequenced by a 16S rRNA gene primer set targeting bacteria and archaea. Findings: A total of 550 samples from 36 mother-neonate pairs with vaginal births and 42 mother-neonate pairs with caesarean sections were included in this study. The negative controls showed that the data analysis in this study was not affected by contamination. There was a high diversity of microbial communities in the pregnancy environment of the foetus. Meconium samples could be divided into three distinct types that were not influenced by the delivery method. Interpretation: The distribution patterns of bacterial communities in the meconium, placenta, and foetal membranes were highly similar and had nothing to do with the mode of delivery. For approximately half of the placental microorganisms, the same sequence could be found in the vaginal, rectal, and oral samples of the mother. Keywords: Meconium, Microbiota, Delivery mode, Archaea

Background: Active immunotherapy is an effective, long-lasting, cheap and safe way to suppress the progression of cancer, however, the key issue is to find the appropriate tumor vaccines. Oncoproteins are up-regulated under various stresses, such as radiation, chemodrugs, targeted drugs, hypoxia, malnutrition and so on to promote the cell growth, suppress apoptosis and alter the micro-environment for survival locally and remotely. Oncoproteins and their immunogenic domain could well serve as tumor vaccines to prime the host active anti-tumor immunity, which might enhance the immune (such as anti-PD-1) modulators given exogenously on effective stage. Methods: Proteomics and bio-information analysis were performed to identify potential tumor associated antigens (TAAs) induced by 8 Gy irradiation (IR). Then, peptides derived from CD151 were designed and synthesized according to MHC I binding and immunogenicity. Cell killing assay, flow cytometer, immunohistochemistry staining, and in vivo bioluminescence imaging were applied to assessed active anti-tumor immunity triggered by CD151 peptides in H22 hepatoma primary tumor and experimental 4T1 breast cancer lung metastasis model. Results: We utilized 8 Gy irradiation as therapeutic stress to up-regulate a panel of oncoproteins, and identified CD151 as a TAA according to proteomics and bio-information analysis. Two CD151 peptides were synthesized as "tumor vaccine" to immunize the mice, which triggered active anti-cancer immunity against both the primary growth of H22 hepatoma and the lung metastasis of 4T1 breast cancer in two mouse model via activation of CD8+IFNγ+ lymphocytes and their targeted cytotoxicity as well as reduction of negative regulator of MDSC cells. The survival of mice with lung metastases in CD151 peptide immunized group was also prolonged. Conclusions: The stress-upregulated oncoproteins defined by proteomics and bio-information on 8 Gy irradiated tumor cells are the good candidates for designing immunogenic peptides as tumor vaccines. Anti-tumor active immunity primed by peptides from tetraspanin oncoprotein CD151 could exert effective, long-lasting, cheap and safe way to suppress the progression of cancer. Funding: The project was funded in part by the grants of Fujian Province Natural Science Foundation (2017J01260); the Key Clinical Specialty Discipline Construction Program of Fujian; the National Clinical Key Specialty Construction Program; Fujian Engineering Research Center of Cancer precision Diagnosis and Immunotherapy; Science&Technology Program of Fujian Province, China (#2018Y2003). Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: Animal experiments were approved by Fujian Medical University Institutional Animal Ethical Committee (FJMU IACUC #2018-075). The applicable institutional guidelines for the care and use of animals were followed.

Background: Uncoupling protein 1 (UCP1) is predominantly found in brown adipose tissue mitochondria, and mediates energy dissipation to generate heat rather than ATP via functional mitochondrial uncoupling. However, little is known about its expression and function in kidney. Methods: We carried out a mRNA microarray analysis in mice kidneys with ischemia reperfusion (IR) injury. The most dramatically downregulated gene UCP1 after IR was identified, and its role in generation of mitochondrial reactive oxygen species (ROS) and oxidative stress injury was assessed both in vitro and in vivo. Genetic deletion of UCP1 was used to investigate the effects of UCP1 on ischemia or cisplatin-indued acute kidney injury (AKI) in mice. Findings: UCP1 was located in renal tubular epithelial cells in kidney and downregulated in a time-dependent manner during renal IR. Deletion of UCP1 increased oxidative stress in kidneys and aggravated ischemia or cisplatin induced AKI in mice.Viral-based overexpression of UCP1 reduced mitochondrial ROS generation and apoptosis in hypoxia-treated tubular epithelial cells. Furthermore, UCP1 expression was regulated by peroxisome proliferator-activator receptor (PPAR) γ in kidneys during renal IR. Overexpression of PPAR-γ resembled UCP1-overexpression phenotype in vitro. Treatment with PPAR-γ agonist could induce UCP1 upregulation and provide protective effect against renal IR injury in UCP1+/+mice, but not in UCP1−/−mice. Interpretation: UCP1 protects against AKI likely by suppressing oxidative stress, and activation of UCP1 represents a potential therapeutic strategy for AKI. Fund: National Natural Science Foundation of China grants, Science and Technology Commission of Shanghai. Keywords: Acute kidney injury, Uncoupling protein 1, Reactive oxygen species, Peroxisome proliferator-activator receptor γ

Background The infiltrative nature and lymphatic metastasis of head and neck squamous cell carcinoma (HNSCC) are the main reasons leading to its poor prognosis. Methods A multimodal surface-enhanced resonance Raman spectroscopy (SERRS) and magnetic resonance (MR) nanoprobe, in which paramagnetic chelators and heptamethine cyanine-based Raman reporter molecules were functionalized on a gold nanostar (AuS) surface was developed. Preoperative MRI and intraoperative SERRS-guided surgery were performed on rabbits bearing head and neck VX2 tumours to determine feasibility of the MR/SERRS probe in defining tumour marginal infiltration and lymph nodes metastasis. Findings Preoperative T1-weighted MRI (T1W-MRI) unambiguously delineated the orthotopic head and neck VX2 tumour xenograft and detected the metastatic lymph nodes in rabbit models after intravenous administration of the probe. With the assistance of a hand-held Raman detector, the probe not only intra-operatively demarcated invasive tumour margins but also successfully distinguished metastatic lymph nodes via a remarkable attenuated Raman signal. Importantly, the group of rabbits subjected to the SERRS-guided surgery exhibited prolonged median survival time (78 days) compared with that of the control group without surgical intervention (29 days) or the group treated with conventional white-light-guided surgery (42 days) (P Interpretation we developed a novel AuS-based multimodal MR/SERRS probe. The capability of this probe to identify both a tumour xenograft and metastatic lymph nodes preoperatively by MRI and intra-operatively by SERRS not only avoids the need for unnecessary resection of neurological structures but also provides a new opportunity to improve the surgical prognosis of head and neck carcinoma of infiltrative nature.

Background: Rasal2 has diametric effects on progression of oestrogen receptor-positive (ER+) and -negative (ER–) breast cancers. The relevant causes are unknown. It is also unknown whether the effects of Rasal2 are mediated by an exosome-transport process. Methods: Exosomes were purified from breast cancer cells and identified by transmission electron microscopy and flow cytometry analysis. In vivo and in vitro experiments were conducted to investigate the role of Rasal2 in exosome-mediated breast cancer progression. Western blot analysis was performed to detect Rasal2 and p-Rasal2 (phosphorylated Rasal2) expression in ER+/ER− breast cancer cells and in exosomes, cancer tissues and blood of patients with ER+ or ER− breast cancer. Findings: Phosphorylation of Rasal2 at Serine 237 promoted tumour growth in both ER+ and ER− tumour cells and tissues. The functions of both p-Rasal2 and non-p-Rasal2 (non-phosphorylated-Rasal2) in the modulation of breast cancer progression are exosome-mediated. p-Rasal2 expression in ER+ breast cancer cells and exosomes, cancer tissues and blood was significantly lower than in ER− tumour cells and patients. Interpretation: p-Rasal2 facilitates tumour progression in both ER+ and ER− breast cancers. The ratio of p-Rasal2/non-p-Rasal2 in ER+ and ER− breast cancers is one of the factors deciding the role of Rasal2 (or total Rasal2) as a suppressor in ER+ breast cancers or as a promoter in ER− breast cancers. Targeting the phosphorylation of Rasal2 machinery may therefore be useful as a therapy to restrain breast cancer progression by reducing p-Rasal2/non-p-Rasal2 ratio, especially in ER− breast cancers. Fund: NSFC and Hong Kong Research Grants Council. Keywords: ER+ and ER– breast cancer, Phosphorylation, Phosphorylated Rasal2 (p-Rasal2) and Rasal2, Tumour progression, Exosomal transport

Background: Interleukin-18 (IL-18) is involved in endothelial activation and dysfunction, and in the pathogenesis and severity of acute graft-versus-host disease (aGVHD). Its relevance for patient outcome after allogeneic stem cell transplantation (alloSCT) has not yet been comprehensively addressed. Methods: Pre-transplant serum levels of free IL-18 were retrospectively assessed in a cohort of 589 patients (training cohort). Results were validated in 688 patients allografted in a different centre. The primary endpoint was overall survival (OS). Secondary endpoints included incidences of non-relapse mortality (NRM), relapse, and aGVHD. Findings: In the training cohort, higher pre-transplant levels of free IL-18 were significantly associated with worse OS (hazard ratio [HR] per 1-log2 increase, 1.25, P = 0.008) in multivariable models. This was due to a higher hazard of NRM (HR per 1-log2 increase, 1.39, P = 0.001), rather than relapse. The associations of pre-transplant free IL-18 with higher NRM (HR per 1-log2 increase, 1.24, P = 0.02) and shorter OS (HR per 1-log2 increase, 1.22, P = 0.006) were confirmed in the validation cohort. In both cohorts, the correlations of higher pre-transplant free IL-18 serum levels with increased NRM and worse OS were mainly driven by fatal infectious complications. No associations with incidence of aGVHD were observed. Interpretation: Higher pre-transplant levels of free IL-18 were associated with non-relapse and overall mortality after alloSCT. Our results may provide a rationale for prospective studies evaluating IL-18 status and inhibition of IL-18 activity in patients undergoing allografting. Keywords: Interleukin-18, Allogeneic stem cell transplantation, Outcome, Mortality

Background: Understanding how cells respond to mitotic poisons is of great biomedical and clinical significance. However, it remains unknown how cell-death or survival is determined during exposure to anti-mitotic drugs. Methods: The biological effects of SLC39A6 (LIV-1) and GrpE-like 1 (GRPEL1) on mitotic exit and apoptosis were evaluated both in vitro and in vivo using flow cytometry, western blotting, xenografts and time-lapse imaging. The interactions between proteins and the ubiquitination of GRPEL1 were assessed by GST pull down, immunoprecipitation and mass spectrometry analysis. The expression of LIV-1 in cancers was assessed by immunohistochemistry. Findings: Overexpression of LIV-1 led to direct apoptosis. Depleted for LIV-1 evade anti-mitotic agent-induced killing through a rapid exit from arrested mitosis. LIV-1 interacts with GRPEL1 and Stabilizes GRPEL1 Protein by Preventing Ubiquitylation of GRPEL1. LIV-1-GRPEL1 axis depletion works to reduce the mitotic arrest by inducing PP2A-B55α phosphates activity, while inhibit apoptosis by banding AIF and preventing the latter's release into the nucleus. Loss of function in this axis was frequent in multiple types of human epithelial cancer. Interpretation: These data demonstrate that LIV-1-GRPEL1 axis dually regulates mitotic exit as well as apoptosis by interacting with PP2A B55α and AIF. Its discovery constitutes a conceptual advance for the decisive mechanism of cell fate during damaged mitosis. Fund: National Clinical Research Center for Obstetric and Gynecologic Diseases, the National Natural Science Foundation of China. Keywords: LIV-1-GRPEL1 axis, Mitotic exit, Apoptosis, Anti-mitotic agents, Human cancer

Background: In multiple sclerosis (MS), immune up-regulation is coupled to subnormal immune response to interferon-β (IFN-β) and low serum IFN-β levels. The relationship between the defect in IFN signalling and acute and long-term effects of IFN-β on gene expression in MS is inadequately understood. Methods: We profiled IFN-β-induced transcriptome shifts, using high-resolution microarrays on 227 mononuclear cell samples from IFN-β-treated MS Complete Responders (CR) stable for five years, and stable and active Partial Responders (PR), stable and active untreated MS, and healthy controls. Findings: IFN-β injection induced short-term changes in 1,200 genes compared to baseline expression after 4-day IFN washout. Pre-injection after washout, and in response to IFN-β injections, PR more frequently had abnormal gene expression than CR. Surprisingly, short-term IFN-β induced little shift in Th1/Th17/Th2 gene expression, but up-regulated immune-inhibitory genes (ILT, IDO1, PD-L1). Expression of 8,800 genes was dysregulated in therapy-naïve compared to IFN-β-treated patients. These long-term changes in protein-coding and long non-coding RNAs affect immunity, synaptic transmission, and CNS cell survival, and correct the disordered therapy-naïve transcriptome to near-normal. In keeping with its impact on clinical course and brain repair in MS, long-term IFN-β treatment reversed the overexpression of proinflammatory and MMP genes, while enhancing genes involved in the oligodendroglia-protective integrated stress response, neuroprotection, and immunoregulation. In the rectified long-term signature, 277 transcripts differed between stable PR and CR patients. Interpretation: IFN-β had minimal short-term effects on Th1 and Th2 pathways, but long-term it corrected gene dysregulation and induced immunoregulatory and neuroprotective genes. These data offer new biomarkers for IFN-β responsiveness. Funding: Unrestricted grants from the US National MS Society, NMSS RG#4509A, and Bayer Pharmaceuticals Keywords: Gene expression, ILT, Immune regulation, Interferon-beta, Multiple sclerosis, RNA microarray

Background Blockade of mitotic progression is an ideal approach to induce mitotic catastrophe that suppresses cancer cell expansion. Cdc20 is a critical mitotic factor governing anaphase initiation and the exit from mitosis through recruiting substrates to APC/C for degradation. Results from recent TCGA (The Cancer Genome Atlas) and pathological studies have demonstrated a pivotal oncogenic role for Cdc20-APC/C in tumor progression as well as drug resistance. Thus, deprivation of the mitotic role for Cdc20-APC/C by either inhibition of Cdc20-APC/C activity or elimination of Cdc20 protein via induced protein degradation emerges as an effective therapeutic strategy to control cancer. Methods We designed a proteolysis targeting chimera, called CP5V, which comprises a Cdc20 ligand and VHL binding moiety bridged by a PEG5 linker that induces Cdc20 degradation. We characterized the effect of CP5V in destroying Cdc20, arresting mitosis, and inhibiting tumor progression by measuring protein degradation, 3D structure dynamics, cell cycle control, tumor cell killing and tumor inhibition using human breast cancer xenograft mouse model. Findings Results from our study demonstrate that CP5V can specifically degrade Cdc20 by linking Cdc20 to the VHL/VBC complex for ubiquitination followed by proteasomal degradation. Induced degradation of Cdc20 by CP5V leads to significant inhibition of breast cancer cell proliferation and resensitization of Taxol-resistant cell lines. Results based on a human breast cancer xenograft mouse model show a significant role for CP5V in suppressing breast tumor progression. Interpretation CP5V-mediated degradation of Cdc20 could be an effective therapeutic strategy for anti-mitotic therapy.

Background: Although various molecular subtypes of bladder cancer (BC) have been investigated, most of these studies have focused on muscle-invasive BC (MIBC). A few studies have investigated non-muscle-invasive BC (NMIBC) or NMIBC and MIBC together, but none has classified progressive NMIBC or immune checkpoint inhibitor (ICI)-based therapeutic responses in early-stage BC patients. Methods: A total of 1,934 samples from seven patient cohorts were used. We performed unsupervised hierarchical clustering to stratify patients into distinct subgroups and constructed a classifier by applying SAM/PAM algorithms. We then investigated the association between molecular subtypes and immunotherapy responsiveness using various statistical methods. Findings: We explored large-scale genomic datasets encompassing NMIBC and MIBC, redefining four distinct molecular subtypes, including a subgroup containing progressive NMIBC and MIBC with poor prognosis that would benefit from ICI treatment. This subgroup showed poor progression-free survival with the distinct features of high mutation load, activated cell cycle, and inhibited TGFβ signalling. Importantly, we verified that BC patients with this subtype were significantly responsive to an anti-PD-L1 agent in the IMvigor210 cohort. Interpretation: Our results reveal an immunotherapeutic option for ICI treatment of highly progressive NMIBC and MIBC with poor prognosis. Funding: This research was supported by the National Research Foundation of Korea grant funded by the Korean government, a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health and Welfare, Republic of Korea, and a grant from the KRIBB Research Initiative Program. Keywords: Bladder cancer, Disease progression, Subtype, Immunotherapy, Genomic signature

Background: Monoclonal antibodies (mAbs) targeting negative regulators, or checkpoint molecules (e.g. PD1/PD-L1 & CTLA4), of anti-tumoural T cells have demonstrated clinical efficacy in treating several neoplastic diseases. While many patients enjoy remarkable responses to checkpoint inhibitors, a majority show adverse effects. Understanding how checkpoint inhibitors may augment established chemotherapy or radiotherapy regimens or other immunotherapies like oncolytic viruses may lead to better clinical outcomes measured by improved efficacy with reduced toxicity. Here, we assess how Newcastle disease virus (NDV), an oncolytic virus in clinical testing, may interact with radiotherapy to enhance checkpoint inhibitor blockade. Methods: An immunocompetent B16-F10 murine melanoma model, generally considered to be a poorly immunogenic or “cold” tumour, was utilised to query whether combining localised radiotherapy with NDV may be more effective than either therapy alone in controlling tumours in mice treated with anti-PD1 or anti-CTLA4 monoclonal antibodies. We also investigated whether localised administration of a checkpoint inhibitor through an intratumoural injection of NDV that expresses anti-CTLA4 single-chain variable fragment (scFv) is comparable to systemic administration of anti-CTLA4 when combined with radiation in mediating its anti-tumour efficacy. Response rates were characterised by measuring tumour size over time, observation of complete tumour regression, and overall survival. Findings: Our results show that combining NDV plus radiotherapy with checkpoint inhibitors (PD1 or CTLA4 targeted mAbs) results in significantly better complete tumour regression rates with an abscopal effect in a murine model of melanoma than either single therapy combined with checkpoint inhibitors. Finally, we also show that localised administration of a recombinant NDV expressing anti-CTLA4 plus radiation is comparable to systemic anti-CTLA4 plus radiation in mediating its anti-tumour effect as assayed by survival benefit. Interpretation: Our results show that oncolytic NDV plus radiotherapy work together with checkpoint inhibitors to enhance tumour clearance of murine melanoma. NDV is an effective radiotherapy dose-sparing and immunotherapeutic agent capable of transgenic, in vivo expression of an anti-CTLA4 targeted scFv antibody with the potential to spare systemic exposure. Funding: The National Institutes of Health grant HHSN272201400008C supported the work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Keywords: Oncolytic viruses, Cancer immunotherapy, Newcastle disease virus, Radiation therapy, Checkpoint inhibitors, Radiosensitiser, Radioenhancer

Summary: Background: Bronchiolitis obliterans syndrome (BOS) after allo-HSCT is a devastating complication with limited therapeutic options. We aimed to assess the efficacy and safety of mesenchymal stem cells (MSCs) in BOS after allo-HSCT. Methods: This multicenter prospective cohort study enrolled 81 allo-HSCT recipients whose BOS were diagnosed within 6 months. The choice of prednisone and azithromycin combined with or without MSCs was based on patient preferences (MSC n = 49, non-MSC n = 32). The primary endpoint was response rate at 3 months, defined as the proportion of patients achieving FEV1 improvement or steroid sparing. The trial was registered at ClinicalTrials.gov (NCT02543073). Findings: Response rate was 35/49 patients (71%, 95% CI 59 to 84%) and 14/32 (44%, 27 to 61%) in MSC and non-MSC group, respectively (p = 0.013). The addition of MSCs was associated with a better difference for change in FEV1 rate of decline, compared to non-MSC group (53 mL/months, 2 to 103; p = 0.040). The 3-year overall survival post-diagnosis was 70.6% (55.9 to 85.3%) and 58.2% (36.1 to 78.5%) in MSC and non-MSC group, respectively (p = 0.21). Clinical improvement was accompanied by a significant increase of interleukin (IL)-10-producing CD5+B cells. There was no statistical difference in the rates of infections and leukemia relapse between the two groups. MSCs were well-tolerated with no serious adverse events. Interpretation: MSCs offer an effective and safe therapeutic option for BOS after allo-HSCT. Our study strengthens evidence for clinical use of MSC therapy in BOS. These data also provide novel insight into potential biological mechanisms of MSC treatment and support further investigation in larger randomized controlled trials. Funding: National Key R&D Program of China, National Natural Science Foundation of China, Health Collaborative Innovation Major Projects of Guangzhou City, Science and Technology Planning Project of Guangdong Province. Keywords: Allogeneic hematopoietic stem cell transplantation, Chronic graft-versus host disease, Bronchiolitis obliterans syndrome, Mesenchymal stem cells

Summary: Background: Ebola virus (EBOV), variant Makona, was the causative agent of the 2013–2016 West African epidemic responsible for almost 30,000 human infections and over 11,000 fatalities. During the epidemic, the development of several experimental vaccines was accelerated through human clinical trials. One of them, the vesicular stomatitis virus (VSV)-based vaccine VSV-EBOV, showed promising efficacy in a phase 3 clinical trial in Guinea and is currently used in the ongoing EBOV outbreak in the northeastern part of the Democratic Republic of the Congo (DRC). This vaccine expresses the EBOV-Kikwit glycoprotein from the 1995 outbreak as the immunogen. Methods: Here we generated a VSV-based vaccine expressing the contemporary EBOV-Makona glycoprotein. We characterized the vaccine in tissue culture and analyzed vaccine efficacy in the cynomolgus macaque model. Subsequently, we determined the dose-dependent protective efficacy in nonhuman primates against lethal EBOV challenge. Findings: We observed complete protection from disease with VSV-EBOV doses ranging from 1 × 107 to 1 × 101 plaque-forming units. Some protected animals receiving lower vaccine doses developed temporary low-level EBOV viremia. Control animals developed classical EBOV disease and reached euthanasia criteria within a week after challenge. This study demonstrates that very low doses of VSV-EBOV uniformly protect macaques against lethal EBOV challenge. Interpretation: Our study provides missing pre-clinical data supporting the use of reduced VSV-EBOV vaccine doses without decreasing protective efficacy and at the same time increase vaccine safety and availability - two critical concerns in public health response. Funding: Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. Keywords: Ebola virus, EBOV-Makona, Macaque model, VSV-EBOV, Low-dose vaccination

Background: Although bone morphogenetic protein 6 (BMP6) signaling pathway has been implicated in many types of cancer, its role of tumorigenesis seems to be controversial and its ubiquitin-modifying mechanisms have not been fully addressed. Our study was designed to investigate how BMP6 signaling pathway is regulated by ubiquitin-modifying systems and to address molecular and clinical significance in colorectal cancers. Methods: Human deubiquitnase (DUB) siRNA library was used to screen the specific DUB, named PSMD14, involved in BMP6 signaling pathway. Immunoblot, immunoprecipitation and ubiquitination assays were used to analyze targets of the PSMD14. A role of PSMD14-mediated BMP6 signaling pathway for malignant cancer progression was investigated using in vitro and in vivo model of colorectal cancers as well as clinical samples of colorectal cancer patients. Findings: The deubiquitinase PSMD14 acts as a positive regulator for the initiation of the BMP6 signaling pathway through deubiquitinating K48-linked ALK2 type I receptor ubiquitination mediated by Smurf1 E3 ligase, resulting in increased stability of the ALK2. This role of PSMD14 is independent of its intrinsic role in the 26S proteasome system. Furthermore, either PSMD14 or ALK2 depletion significantly decreases tumorigenesis of HCT116 colorectal cancer cells in a xenograft model as well as cancer stemness/chemoresistance, and expression of the PSMD14 and ALK2 gene are correlated with malignant progression and the survival of colorectal cancer patients. Interpretation: These findings suggest that the PSMD14-ALK2 axis plays an essential role in initiation of the BMP6 signaling pathway and contributes to tumorigenesis and chemoresistance of colorectal cancers. Key words: PSMD14, Deubiqutinase, ALK2 receptor, Smurf1, BMP6, Colorectal cancers

Background: No effective approaches to target mutant Kras have yet been developed. Immunoprevention using KRAS-specific antigenic peptides to trigger T cells capable of targeting tumor cells relies heavily on lipid metabolism. To facilitate better TCR/peptide/MHC interactions that result in better cancer preventive efficacy, we combined KVax with avasimibe, a specific ACAT1 inhibitor, tested their anti-cancer efficacy in mouse lung cancer models, where Kras mutation was induced before vaccination. Methods: Control of tumor growth utilizing a multi-peptide Kras vaccine was tested in combination with avasimibe in a syngeneic lung cancer mouse model and a genetically engineered mouse model (GEMM). Activation of immune responses after administration of Kras vaccine and avasimibe was also assessed by flow cytometry, ELISpot and IHC. Findings: We found that Kras vaccine combined with avasimibe significantly decreased the presence of regulatory T cells in the tumor microenvironment and facilitated CD8+ T cell infiltration in tumor sites. Avasimibe also enhanced the efficacy of Kras vaccines target mutant Kras. Whereas the Kras vaccine significantly increased antigen-specific intracellular IFN-γ and granzyme B levels in CD8+ T cells, avasimibe significantly increased the number of tumor-infiltrating CD8+ T cells. Additionally, modulation of cholesterol metabolism was found to specifically impact in T cells, and not in cancer cells. Interpretation: Avasimibe complements the efficacy of a multi-peptide Kras vaccine in controlling lung cancer development and growth. This treatment regimen represents a novel immunoprevention approach to prevent lung cancer. Keywords: Chemoimmunoprevention, Kras, Peptide vaccine, Avasimibe, Cholesterol metabolism, T cell

Background Cirrhosis-associated immune dysfunction (CAID) contributes to high sepsis risk in patients with chronic liver disease. Various innate and; to a lesser extent; adaptive immune dysfunctions have been described as contributors to CAID leading to immune-paresis and impaired anti-microbial response in cirrhosis. In this study, we examined the phenotype of CD8+ T cells in chronic liver disease with the aim to evaluate changes that might contribute to impaired immune responses. Methods Sixty patients with cirrhosis were prospectively recruited for this study. CD8+ T cells from peripheral blood, ascites and liver explants were characterized using flow cytometry and immunohistochemistry, respectively. The transcriptional signature of flow-sorted HLA-DR+CD8+ T cells was performed using Nanostring™ technology. HLA-DR+CD8+ T cells interactions with PBMCs and myeloid cells were tested in vitro. Findings Peripheral CD8+ T cells from cirrhotic patients displayed an altered phenotype characterized by high HLA-DR and TIM-3 surface expression associated with concomitant infections and disease severity, respectively. Paired peritoneal CD8+ T cells expressed more pronounced levels of HLA-DR and PD-1 compared to peripheral CD8+ T cells. HLA-DR+CD8+ T cells were enriched in cirrhotic livers compared to controls. TIM-3, CTLA-4 and PD-1 levels were highly expressed on HLA-DR+CD8+ T cells and co-expression of HLA-DR and PD1 was higher in patients with poor disease outcomes. Genes involved in cytokines production and intracellular signalling pathways were strongly down-regulated in HLA-DR+CD8+ T cells. In comparison to their HLA-DR− counterparts, HLA-DR+CD8+ T cells promoted less proliferation of PBMCs and induced phenotypic and functional dysfunctions in monocytes and neutrophils in vitro. Interpretation In patients with cirrhosis, CD8+ T cells display a phenotypic, functional and transcriptional profile which may contribute to CAID. Fund This work was supported by Medical Research Council, the Rosetrees Charitable Trust, Robert Tournut 2016 grant (Societe Nationale Francaise de GastroEnterologie), Gilead® sciences, and NIHR Imperial Biomedical Research Centre.

Background: Hepatic encephalopathy (HE) is a severe neuropsychiatric syndrome caused by various types of liver failure resulting in hyperammonemia-induced dysfunction of astrocytes. It is unclear whether autophagy, an important pro-survival pathway, is altered in the brains of ammonia-intoxicated animals as well as in HE patients. Methods: Using primary rat astrocytes, a co-culture model of primary mouse astrocytes and neurons, an in vivo rat HE model, and post mortem brain samples of liver cirrhosis patients with HE we analyzed whether and how hyperammonemia modulates autophagy. Findings: We show that autophagic flux is efficiently inhibited after administration of ammonia in astrocytes. This occurs in a fast, reversible, time-, dose-, and ROS-dependent manner and is mediated by ammonia-induced changes in intralysosomal pH. Autophagic flux is also strongly inhibited in the cerebral cortex of rats after acute ammonium intoxication corroborating our results using an in vivo rat HE model. Transglutaminase 2 (TGM2), a factor promoting autophagy, is upregulated in astrocytes of in vitro- and in vivo-HE models as well as in post mortem brain samples of liver cirrhosis patients with HE, but not in patients without HE. LC3, a commonly used autophagy marker, is significantly increased in the brain of HE patients. Ammonia also modulated autophagy moderately in neuronal cells. We show that taurine, known to ameliorate several parameters caused by hyperammonemia in patients suffering from liver failure, is highly potent in reducing ammonia-induced impairment of autophagic flux. This protective effect of taurine is apparently not linked to inhibition of mTOR signaling but rather to reducing ammonia-induced ROS formation. Interpretation: Our data support a model in which autophagy aims to counteract ammonia-induced toxicity, yet, as acidification of lysosomes is impaired, possible protective effects thereof, are hampered. We propose that modulating autophagy in astrocytes and/or neurons, e.g. by taurine, represents a novel strategy to treat liver diseases associated with HE. Funding: Supported by the DFG, CRC974 “Communication and Systems Relevance in Liver Injury and Regeneration“, Düsseldorf (Project number 190586431) Projects A05 (DH), B04 (BG), B05 (NK), and B09 (ASR). Keywords: Hepatic encephalopathy, Liver disease, Autophagy, Lysosome, Taurine

Background: Identifying populations at high risk of HIV transmission is critical for prioritizing treatment and prevention resources and achieving the UNAIDS 90-90-90 Targets. Methods: HIV transmission rates can be estimated from phylogenetic trees as viral lineage-level diversification rates. To identify HIV-1 transmission foci in British Columbia, Canada, we inferred diversification rates from phylogenetic trees of 36 271 HIV-1 sequences from 9630 anonymized individuals. Diversification rates were combined with sociodemographic and clinical data, then aggregated by patients’ area of residence to predict the distribution of new HIV cases between 2008 and 2018. The predictive power of the model was compared with a phylogenetically uninformed model. Findings: Aggregated diversification rate measures were predictive of new HIV cases in the subsequent year after adjusting for prevalent and incident cases in the previous year. For every one-unit increase in the mean of the top five diversification rates, the number of new HIV cases increased by on average 1·38-fold (95% CI, 1·28–1·49). In a blind prediction of 2018 cases, diversification rate improved the model's specificity by 12%, accuracy by 9%, top 20 agreement by 100%, and correlation of predicted and observed values by 162% relative to a model that incorporated epidemiological data alone. Interpretation: By predicting the distribution of future HIV cases, a combined phylogenetic and epidemiological approach identifies hotspots where public health resources are needed most. Funding: Canadian Institutes of Health Research, University of British Columbia, Public Health Agency of Canada, Genome Canada, Genome BC, Michael Smith Foundation for Health Research, and BC Centre for Excellence in HIV/AIDS. Keywords: HIV, Phylogenetics, Epidemiology, Transmission, Predictive model

Background Sjogren's syndrome (SS) is one of the most common autoimmune disorders leading to exocrine gland dysfunction. Both immune-dependent processes – like Type I Interferon (IFN) signaling and immune-independent processes – such as calcium signaling in epithelial cells – contribute to disease pathophysiology. However, a mechanistic link between these processes has not been demonstrated. Methods Primary human salivary gland cells were used to evaluate the differential expression of miRNAs with smRNA-seq in primary epithelial cells culture and digital PCR was conducted in SS human salivary glands (SG) biopsies to verify the results. With siRNA screening and pull-down assays to establish the role of miRNA in IFN activation. Findings Activation of IFN-β by miR-1248 is through the direct association with both RIG-I and AGO2. Further functional studies establish a unique dual functional role of miR-1248 in phSG cells: i) activation of the RIG-I pathway by acting as ligand of this sensor leading to IFN production and ii) regulation of the expression of mRNAs through the canonical microRNA function. Importantly, ITPR3, a key component of calcium signaling in epithelial cells, that has previously shown to be downregulated in SS SG, was directly targeted and downregulated by miR-1248, inducing the same functional calcium signaling changes as observed in SS SGs. Interpretation Identification of the first endogenous mammalian microRNA that binds to RIG-I inducing IFN production but also demonstrate a novel pathophysiological underlying mechanism in which miR-1248 overexpression links two major pathways associated with SS, namely activation of IFN production with modulation of calcium signaling. Together, these findings suggest a unifying hypothesis for the immune-independent and -dependent processes contributing to the pathogenesis of SS. Fund This research was supported by the Intramural Research Program of the National Institutes of Health (NIH), National Institute of Dental and Craniofacial Research (NIDCR).

Background: Non-ischemic dilated cardiomyopathy (NIDCM) responds variably to intramyocardial injection of mesenchymal stem cells (MSCs). We hypothesized that NIDCM genotype may influence responsiveness to MSC therapy and performed genotyping on all patients in the POSEIDON-DCM trial. Methods: POSEIDON-DCM patients (n = 34) underwent genetic sequence analysis and deletion/duplication testing. The results were classified as positive for pathological variants (PV+; n = 8), negative for any variants (V−; n = 6), or as variants of uncertain significance (VUS; n = 20). All outcomes of therapy were analysed for each category of genetic results. Findings: The 3 groups were indistinguishable at baseline with regard to ejection fraction (EF), demographics, medication use, or functional parameters. V− patients had an increase in EF at 12 months: +13.6% (IQR = +7.8%; +20.5%; p = 0.002), compared with VUS (+6.5%; IQR = +0.9%, +11.1%; p = 0.005) and PV+(−5.9%; IQR = −12.7%, +1.0; p = 0.2; p = 0.01 between groups). Six-minute walk distance improved in V- patients, but not in VUS and PV+. V− patients improved MLHFQ, compared to the other 2 groups, which did not improve over time. EPCCFUs increased by 9.7 ± 1.9 in V− (p = 0.009) compared to VUS and PV+ patients. V− patients had one-year survival (100%) compared with VUS (85%) and PV+ (40%; p = 0.015 log-rank). Similarly, MACE rates were lower in V− (0%) than PV+ (61.9%) or VUS (42.2%; p = 0.021 log-rank). Interpretation: Our findings support the concept that the genetic profile of NIDCM patients plays a role in responsiveness to MSC therapy, with V− patients more likely to benefit and the converse for PV+. This observation emphasizes the need for further genetic studies, because of important implications for the management of NIDCM syndromes. Keywords: Precision medicine, Variants of uncertain significance, Positive for pathologic/likely pathologic variant, Dilated cardiomyopathy

Background: Urea, the end product of protein metabolism, has been considered to have negligible toxicity for a long time. Our previous study showed a depression phenotype in urea transporter (UT) B knockout mice, which suggests that abnormal urea metabolism may cause depression. The purpose of this study was to determine if urea accumulation in brain is a key factor causing depression using clinical data and animal models. Methods: A meta-analysis was used to identify the relationship between depression and chronic diseases. Functional Magnetic Resonance Imaging (fMRI) brain scans and common biochemical indexes were compared between the patients and healthy controls. We used behavioural tests, electrophysiology, and molecular profiling techniques to investigate the functional role and molecular basis in mouse models. Findings: After performing a meta-analysis, we targeted the relevance between chronic kidney disease (CKD) and depression. In a CKD mouse model and a patient cohort, depression was induced by impairing the medial prefrontal cortex. The enlarged cohort suggested that urea was responsible for depression. In mice, urea was sufficient to induce depression, interrupt long-term potentiation (LTP) and cause loss of synapses in several models. The mTORC1-S6K pathway inhibition was necessary for the effect of urea. Lastly, we identified that the hydrolysate of urea, cyanate, was also involved in this pathophysiology. Interpretation: These data indicate that urea accumulation in brain is an independent factor causing depression, bypassing the psychosocial stress. Urea or cyanate carbamylates mTOR to inhibit the mTORC1-S6K dependent dendritic protein synthesis, inducing impairment of synaptic plasticity in mPFC and depression-like behaviour. CKD patients may be able to attenuate depression only by strict management of blood urea. Keywords: Urea, Depression, LTP, Carbamylation, mTOR

Background SWELL1 was recently demonstrated to be an indispensable part of the volume-regulated anion channel (VRAC). VRAC is reported to participate in cell proliferation, survival, and migration. However, the correlation between SWELL1 and hepatocellular carcinoma (HCC) remains poorly-understood. In this study, we tried to explore the role of SWELL1 in HCC. Methods Immunohistochemistry and quantitative real-time-PCR (qRT-PCR) was used to measure SWELL1 expression in HCC samples obtained from patients with HCC. The effects of SWELL1 on HCC cell proliferation, apoptosis, and metastasis were analysed by corresponding cytological experiments including Cell Counting Kit-8 (CCK8), colony-forming, 5-ethynyl-2′-deoxyuridine (EdU), cell cycle analysis, TUNEL, Annexin V and PI staining, wound healing, transwell, and so on. BALB/c nude mice were used for the in vivo assays. qRT-PCR and western blotting was performed for molecular mechanisms. Findings SWELL1 was highly expressed in HCC tissues, and related to the poor prognosis. In vitro, the over-expression of SWELL1 significantly induced cell proliferation and migration, and inhibited apoptosis, whereas suppressing SWELL1 had the opposite effects. Moreover, knockdown of SWELL1 suppressed the growth and metastasis of HCC in vivo. Further experiments revealed that SWELL1 induced cell growth by activating the cyclinD1/CDK2 pathway via the connection with PKCa at the signalling level, and regulated cell migration through the JNK pathway in HCC. Interpretation SWELL1 acts as a promoter in the growth and metastasis of HCC cells and may be a potential intervention target for HCC. Fund This work is supported by the National Natural Science Foundation of China (No. 81572422 , 81700515 ).

Background: The development of a clinically useful fibroblast growth factor 21 (FGF21) hormone has been impeded by its inherent instability and weak FGF receptor (FGFR) binding affinity. There is an urgent need for innovative approaches to overcome these limitations. Methods: We devised a structure-based chimerisation strategy in which we substituted the thermally labile and low receptor affinity core of FGF21 with an HS binding deficient endocrinised core derived from a stable and high receptor affinity paracrine FGF1 (FGF1ΔHBS). The thermal stability, receptor binding ability, heparan sulfate and βKlotho coreceptor dependency of the chimera were measured using a thermal shift assay, SPR, SEC-MALS and cell-based studies. The half-life, tissue distribution, glucose lowering activity and adipose tissue remodeling were analyzed in normal and diabetic mice and monkeys. Findings: The melting temperature of the engineered chimera (FGF1ΔHBS-FGF21C-tail) increased by ∼22 °C relative to wild-type FGF21 (FGF21WT), and resulted in a ∼5-fold increase in half-life in vivo. The chimera also acquired an ability to bind the FGFR1c isoform – the principal receptor that mediates the metabolic actions of FGF21 – and consequently was dramatically more effective than FGF21WT in correcting hyperglycemia and in ameliorating insulin resistance in db/db mice. Our chimeric FGF21 also exerted a significant beneficial effect on glycemic control in spontaneous diabetic cynomolgus monkeys. Interpretation: Our study describes a structure-based chimerisation approach that effectively mitigates both the intrinsically weak receptor binding affinities and short half-lives of endocrine FGFs, and advance the development of the FGF21 hormone into a potentially useful drug for Type 2 diabetes. Keywords: Fibroblast growth factor 21, Chimerisation, Thermal stability, Receptor binding affinity, Type 2 diabetes

Background: Thymidylate synthase (TYMS) is a successful chemotherapeutic target for anticancer therapy. Numerous TYMS inhibitors have been developed and used for treating gastrointestinal cancer now, but they have limited clinical benefits due to the prevalent unresponsiveness and toxicity. It is urgent to identify a predictive biomarker to guide the precise clinical use of TYMS inhibitors. Methods: Genome-scale CRISPR-Cas9 knockout screening was performed to identify potential therapeutic targets for treating gastrointestinal tumours as well as key regulators of raltitrexed (RTX) sensitivity. Cell-based functional assays were used to investigate how MYC regulates TYMS transcription. Cancer patient data were used to verify the correlation between drug response and MYC and/or TYMS mRNA levels. Finally, the role of NIPBL inactivation in gastrointestinal cancer was evaluated in vitro and in vivo. Findings: TYMS is essential for maintaining the viability of gastrointestinal cancer cells, and is selectively inhibited by RTX. Mechanistically, MYC presets gastrointestinal cancer sensitivity to RTX through upregulating TYMS transcription, supported by TCGA data showing that complete response cases to TYMS inhibitors had significantly higher MYC and TYMS mRNA levels than those of progressive diseases. NIPBL inactivation decreases the therapeutic responses of gastrointestinal cancer to RTX through blocking MYC. Interpretation: Our study unveils a mechanism of how TYMS is transcriptionally regulated by MYC, and provides rationales for the precise use of TYMS inhibitors in the clinic. Funding: This work was financially supported by grants of NKRDP (2016YFC1302400), STCSM (16JC1406200), NSFC (81872890, 81322034, 81372346) and CAS (QYZDB-SSW-SMC034, XDA12020210). Keywords: Thymidylate synthase, Raltitrexed, MYC, NIPBL, Genome-scale CRISPR-Cas9 knockout screening

Background Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-related tumor. The role of EBV-encoding miR-BART22 is still unclear in NPC. This study aimed to identify the detailed mechanisms by which EBV-miR-BART22 functions as a tumor-promoting factor and evaluate the action of cinobufotalin in treating EBV-miR-BART22-overexpressing NPC cells. Methods Using real-time PCR, western blotting, immunohistochemistry, and In situ hybridization, we detected the expression of miR-BART22 and MAP2K4 in tissues and cells, as well as evaluated their clinical relevance in NPC patients. The effects of miR-BART22 on cell metastasis, stemness and DDP chemoresistance were examined by sphere formation assay, side population analysis, transwell, boyden, in vivo xenograft tumor mouse model et al. Western blotting, immunofluorescence staining, luciferase reporter assay, ChIP, EMSA and Co-IP assay et al. were performed to explore the detailed molecular mechanism of EBV-miR-BART22 in NPC. Finally, we estimated the effects and molecular basis of Cinobufotalin on EBV-miR-BART22-overexpressing NPC cells in vitro and in vivo assays. Findings We observed that EBV-miR-BART22 not only promoted tumor stemness and metastasis, but also enhanced the resistance to Cisplatin (DDP) in vitro and in vivo. Mechanistic analysis indicated that EBV-miR-BART22 directly targeted the MAP2K4 and upregulated non-muscle myosin heavy chain IIA (MYH9) expression by PI3K/AKT/c-Jun-induced transcription. Further, MYH9 interacted with glycogen synthase 3β(GSK3β) protein and induced its ubiquitin degradation by activating PI3K/AKT/c-Jun-induced ubiquitin transcription and the latter combined with increased TRAF6 E3 ligase, which further bound to GSK3β protein. Reductions in the GSK3β protein thus promoted β-catenin expression and nuclear translocation, which induced tumor stemness and the epithelial-to-mesenchymal transition (EMT) signals. Furthermore, we observed that cinobufotalin, a new chemically synthesized compound, significantly suppressed EBV-miR-BART22-induced DDP chemoresistance by upregulating MAP2K4 to suppress MYH9/GSK3β/β-catenin and its downstream tumor stemness and EMT signals in NPC. Finally, clinical data revealed that increased miR-BART22 and reduced MAP2K4 expression caused the poor prognoses of NPC patients. Interpretation Our study provides a novel mechanism that cinobufotalin reversed the DDP chemoresistance and EMT induced by EBV-miR-BART22 in NPC., Graphical abstract Unlabelled Image

Melanoma is known to be a heterogeneous tumor, but the impact of this heterogeneity upon therapeutic response is not well understood. In the current study, we used single cell mRNA analysis and our Single Cell HETerogeneity (SinCHET) platform to define the transcriptional diversity of melanoma and its dynamic response to both BRAF inhibitor therapy and treatment holidays. Our analyses showed melanoma cell lines and patient specimens to be composed of >3 co-existent, transcriptionally distinct states. Among these, State #1 had increased expression of cyclin D1, ERBB3, STAT3/5, MITF and I²-catenin and lower expression of c-JUN and RTKs such as Axl and EGFR. State #2 was characterized by higher expression of ERBB3, Axl and the transcription factor c-JUN. State #3 was characterized by high Axl, c-JUN, E2F1, WEE1, c-MET and EGFR expression and lower expression of MITF, ERBB3 and SMAD9. The cell state composition was dynamically regulated in response to BRAF inhibitor therapy, with State #1 declining and States #2 and #3 increasing in response to drug. The percentage of cells in State #1 recovered following a drug holiday, allowing for a successful therapy rechallenge. Other melanomas that lacked State #1 had varying degrees of intrinsic drug resistance and were not amenable to rechallenge. We next leveraged the differences in fitness between the different transcriptional states in the absence and presence of drug to develop a mathematical model that optimized therapy schedules to retain the drug sensitive population. In vivo validation of the mathematical model demonstrated that the personalized adaptive dosing schedules were better at suppressing tumor growth than either continuous or fixed intermittent BRAF inhibitor schedules. Together our studies provide the first preclinical evidence that transcriptional heterogeneity at the single cell level predicts for the initial sensitivity to BRAF inhibitor therapy. We further demonstrate that manipulating transcriptional heterogeneity through personalized adaptive therapy schedules that can delay the time to resistance. Funding Statement: Supported by SPORE grant P50 CA168536, R21 CA198550, R21 CA216756 (to KSMS) K99 CA226679 (to IS), Moffitt Cancer Center PSOC, U54 CA193489 (EK & AA) and the Cancer Center Support Grant P30 CA076292 from the NIH (NCI/NIH). We also acknowledge the support Miami Center for AIDS Research (CFAR) at the University of Miami Miller School of Medicine funded by P30AI073961. Declaration of Interests: We have no competing interest to declare. Ethics Approval Statement: All animal experiments were carried out in compliance with ethical regulations and protocols approved by the University of South Florida Institutional Animal Care and Use Committee.

Background Aberrant expression of p53 and its downstream gene p21 is closely related to alterations in cell cycle and cell proliferation, and is common among cancer patients. However, the underlying molecular mechanism has not been fully unravelled. ZER6 is a zinc-finger protein with two isoforms possessing different amino termini (N-termini) in their proteins, p52-ZER6 and p71-ZER6. The biological function of ZER6 isoforms, as well as their potential involvement in tumourigenesis and the regulation of p53 remain elusive. Methods The effect of ZER6 isoforms on p53 and p21 was determined using specific knockdown and overexpression. p52-ZER6 expression in tumours was analysed using clinical specimens, while gene modulation was used to explore p52-ZER6 roles in regulating cell proliferation and tumourigenesis. The mechanism of p52-ZER6 regulation on the p53/p21 axis was studied using molecular biology and biochemical methods. Findings p52-ZER6 was highly expressed in tumour tissues, and was closely related with tumour progression. Mechanistically, p52-ZER6 bound to p53 through a truncated KRAB (tKRAB) domain in its N-terminus and enhanced MDM2/p53 complex integrity, leading to increased p53 ubiquitination and degradation. p52-ZER6-silencing induced G0-G1 phase arrest, and subsequently reduced cell proliferation and tumourigenesis. Intriguingly, this regulation on p53 was specific to p52-ZER6, whereas p71-ZER6 did not affect p53 stability, most likely due to the presence of a HUB-1 domain. Interpretation We identified p52-ZER6 as a novel oncogene that enhances MDM2/p53 complex integrity, and might be a potential target for anti-cancer therapy.

Background: End-induction minimal residual disease (MRD) is the strongest predictor of relapse in paediatric acute lymphoblastic leukaemia (ALL), but an understanding of the biological pathways underlying early treatment response remains elusive. We hypothesized that metabolomic profiling of diagnostic bone marrow plasma could provide insights into the underlying biology of early treatment response and inform treatment strategies for high-risk patients. Methods: We performed global metabolomic profiling of samples from discovery (N = 93) and replication (N = 62) cohorts treated at Texas Children's Hospital. Next, we tested the cytotoxicity of drugs targeting central carbon metabolism in cell lines and patient-derived xenograft (PDX) cells. Findings: Metabolite set enrichment analysis identified altered central carbon and amino acid metabolism in MRD-positive patients from both cohorts at a 5% false discovery rate. Metabolites from these pathways were used as inputs for unsupervised hierarchical clustering. Two distinct clusters were identified, which were independently associated with MRD after adjustment for immunophenotype, cytogenetics, and NCI risk group. Three nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, which reduce glycolytic/TCA cycle activities, demonstrated nanomolar-range cytotoxicity in B- and T-ALL cell lines and PDX cells. Interpretation: This study provides new insights into the role of central carbon metabolism in early treatment response and as a potential targetable pathway in high-risk disease. Funding: American Society of Hematology; Baylor College of Medicine Department of Paediatrics; Cancer Prevention and Research Institute of Texas; the Lynch family; St. Baldrick's Foundation with support from the Micaela's Army Foundation; United States National Institutes of Health. Keywords: Acute lymphoblastic leukaemia, Minimal residual disease, Epidemiology, NAMPT, NAMPT inhibitors

Background Resistance to oxaliplatin-based chemotherapy is a major cause of recurrence in colorectal cancer (CRC) patients. There is increasing evidence indicating that circHIPK3 is involved in the development and progression of tumours. However, little is known about the potential role of circHIPK3 in CRC chemotherapy and its molecular mechanisms in chemoresistance also remain unclear. Methods Quantitative real-time PCR was performed to detect circHIPK3 expression in tissues of 2 cohorts of CRC patients who received oxaliplatin-based chemotherapy. The chemoresistant effects of circHIPK3 were assessed by cell viability, apoptosis, and autophagy assays. The relationship between circHIPK3, miR-637, and STAT3 mRNA was confirmed by biotinylated RNA pull-down, luciferase reporter, and western blot assays. Findings In the pilot study, increased circHIPK3 expression was observed in chemoresistant CRC patients. Functional assays showed that circHIPK3 promoted oxaliplatin resistance, which was dependent on inhibition of autophagy. Mechanistically, circHIPK3 sponged miR-637 to promote STAT3 expression, thereby activating the downstream Bcl-2/beclin1 signalling pathway. A clinical cohort study showed that circHIPK3 was upregulated in tissues from recurrent CRC patients and correlated with tumour size, regional lymph node metastasis, distant metastasis, and survival. Interpretation circHIPK3 functions as a chemoresistant gene in CRC cells by targeting the miR-637/STAT3/Bcl-2/beclin1 axis and might be a prognostic predictor for CRC patients who receive oxaliplatin-based chemotherapy. Funding National Natural Science Foundation of China ( 81301506 ), Shandong Medical and Health Technology Development Project ( 2018WSB20002 ), Shandong Key Research and Development Program ( 2016GSF201122 ), Natural Science Foundation of Shandong Province ( ZR2017MH044 ), and Jinan Science and Technology Development Plan ( 201805084 , 201805003 ).

Background: To validate a novel method for post-transplant surveillance to detect kidney allograft rejection via a characteristic constellation of the urine metabolites alanine, citrate, lactate, and urea investigated by nuclear magnetic resonance (NMR) spectroscopy a first prospective, observational study was performed. Methods: Within the UMBRELLA study 986 urine specimens were collected from 109 consecutively enrolled renal transplant recipients, and metabolite constellations were analyzed. A metabolite rejection score was calculated and compared to histopathological results of corresponding indication and protocol allograft biopsies (n = 206). Findings: The metabolite constellation was found to be a useful biomarker to non-invasively detect acute allograft rejection (AUC = 0.75; 95% confidence interval (CI) 0.68–0.83; based on 46 cases and 520 control samples). Combined analysis of the metabolite rejection score and the estimated glomerular filtration rate (eGFR) at the time of urine sampling further improved the overall test performance significantly (AUC = 0.84; 95% CI 0.76–0.91; based on 42 cases and 468 controls). Regarding the time course analysis in patients without rejection episodes the test results remained well below a diagnostic threshold associated with high risk of acute rejection. In other cases, a marked increase above this threshold indicated acute allograft rejection already six to ten days before diagnostic renal biopsies were performed. Interpretation: A combination of an NMR-based urine metabolite analysis and eGFR is promising as a non-invasive test for post-transplant surveillance and to support decision making whether renal allografts need histopathological evaluation. Keywords: Kidney transplant rejection, Urinary metabolites, Biomarker, NMR-spectroscopy, Non-invasive test