Chimeric RNAs reveal putative neoantigen peptides for developing tumor vaccines for breast cancer.

Introduction: We present here a strategy to identify immunogenic neoantigen candidates from unique amino acid sequences at the junctions of fusion proteins which can serve as targets in the development of tumor vaccines for the treatment of breastcancer. Method: We mined the sequence reads of breast tumor tissue that are usually discarded as discordant paired-end reads and discovered cancer specific fusion transcripts using tissue from cancer free controls as reference. Binding affinity predictions of novel peptide sequences crossing the fusion junction were analyzed by the MHC Class I binding predictor, MHCnuggets. CD8+ T cell responses against the 15 peptides were assessed through in vitro Enzyme Linked Immunospot (ELISpot). Results: We uncovered 20 novel fusion transcripts from 75 breast tumors of 3 subtypes: TNBC, HER2+, and HR+. Of these, the NSFP1-LRRC37A2 fusion transcript was selected for further study. The 3833 bp chimeric RNA predicted by the consensus fusion junction sequence is consistent with a read-through transcription of the 5'-gene NSFP1-Pseudo gene NSFP1 (NSFtruncation at exon 12/13) followed by trans-splicing to connect withLRRC37A2 located immediately 3' through exon 1/2. A total of 15 different 8-mer neoantigen peptides discovered from the NSFP1 and LRRC37A2 truncations were predicted to bind to a total of 35 unique MHC class I alleles with a binding affinity of IC50<500nM.); 1 of which elicited a robust immune response. Conclusion: Our data provides a framework to identify immunogenic neoantigen candidates from fusion transcripts and suggests a potential vaccine strategy to target the immunogenic neopeptides in patients with tumors carrying the NSFP1-LRRC37A2 fusion.

Integrative metabolomics and transcriptomics analysis reveals novel therapeutic vulnerabilities in lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) comprises the majority (~85%) of all lung tumors, with lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) being the most frequently diagnosed histological subtypes. Multi-modal omics profiling has been carried out in NSCLC, but no studies have yet reported a unique metabolite-related gene signature and altered metabolic pathways associated with LUAD and LUSC. METHODS: We integrated transcriptomics and metabolomics to analyze 30 human lung tumors and adjacent noncancerous tissues. Differential co-expression was used to identify modules of metabolites that were altered between normal and tumor. RESULTS: We identified unique metabolite-related gene signatures specific for LUAD and LUSC and key pathways aberrantly regulated at both transcriptional and metabolic levels. Differential co-expression analysis revealed that loss of coherence between metabolites in tumors is a major characteristic in both LUAD and LUSC. We identified one metabolic onco-module gained in LUAD, characterized by nine metabolites and 57 metabolic genes. Multi-omics integrative analysis revealed a 28 metabolic gene signature associated with poor survival in LUAD, with six metabolite-related genes as individual prognostic markers. CONCLUSIONS: We demonstrated the clinical utility of this integrated metabolic gene signature in LUAD by using it to guide repurposing of AZD-6482, a PI3Kß inhibitor which significantly inhibited three genes from the 28-gene signature. Overall, we have integrated metabolomics and transcriptomics analyses to show that LUAD and LUSC have distinct profiles, inferred gene signatures with prognostic value for patient survival, and identified therapeutic targets and repurposed drugs for potential use in NSCLC treatment.

Direct reprogramming of cardiomyocytes into cardiac Purkinje-like cells.

Currently, there are no treatments that ameliorate cardiac cell death, the underlying basis of cardiovascular disease. An unexplored cell type in cardiac regeneration is cardiac Purkinje cells; specialized cells from the cardiac conduction system (CCS) responsible for propagating electrical signals. Purkinje cells have tremendous potential as a regenerative treatment because they may intrinsically integrate with the CCS of a recipient myocardium, resulting in more efficient electrical conduction in diseased hearts. This study is the first to demonstrate an effective protocol for the direct reprogramming of human cardiomyocytes into cardiac Purkinje-like cells using small molecules. The cells generated were genetically and functionally similar to native cardiac Purkinje cells, where expression of key cardiac Purkinje genes such as CNTN2, ETV1, PCP4, IRX3, SCN5a, HCN2 and the conduction of electrical signals with increased velocity was observed. This study may help to advance the quest to finding an optimized cell therapy for heart regeneration.

HepT1-derived murine models of high-risk hepatoblastoma display vascular invasion, metastasis, and circulating tumor cells.

Hepatoblastoma (HB) is the most common pediatric primary liver malignancy, and survival for high-risk disease approaches 50%. Mouse models of HB fail to recapitulate hallmarks of high-risk disease. The aim of this work was to generate murine models that show high-risk features including multifocal tumors, vascular invasion, metastasis, and circulating tumor cells (CTCs). HepT1 cells were injected into the livers or tail veins of mice, and tumor growth was monitored with magnetic resonance and bioluminescent imaging. Blood was analyzed with fluorescence-activated cell sorting to identify CTCs. Intra- and extra-hepatic tumor samples were harvested for immunohistochemistry and RNA and DNA sequencing. Cell lines were grown from tumor samples and profiled with RNA sequencing. With intrahepatic injection of HepT1 cells, 100% of animals grew liver tumors and showed vascular invasion, metastasis, and CTCs. Mutation profiling revealed genetic alterations in seven cancer-related genes, while transcriptomic analyses showed changes in gene expression with cells that invade vessels. Tail vein injection of HepT1 cells resulted in multifocal, metastatic disease. These unique models will facilitate further meaningful studies of high-risk HB. This article has an associated First Person interview with the first author of the paper.

Curcumin Reduces Adipose Tissue Inflammation and Alters Gut Microbiota in Diet-Induced Obese Male Mice.

SCOPE: Obesity prevalence continues to increase and contribute to metabolic diseases, potentially by driving systemic inflammation. Curcumin is an anti-inflammatory spice with claimed health benefits. However, mechanisms by which curcumin may reduce obesity-associated inflammation are poorly understood; thus, it is hypothesized that benefits of curcumin consumption may occur through reduced white adipose tissue (WAT) inflammation and/or beneficial changes in gut bacteria. METHODS AND RESULTS: Male B6 mice are fed high-fat diets (HFD, 45% kcal fat) or HFD supplemented with 0.4% (w/w) curcumin (HFC) for 14 weeks. Curcumin supplementation significantly reduces adiposity and total macrophage infiltration in WAT, compared to HFD group, consistent with reduced mRNA levels of M1 (Cd80, Cd38, Cd11c) and M2 (Arginase-1) macrophage markers. Moreover, curcumin supplementation reduces expression of other key pro-inflammatory genes, such as NF-κB p65 subunit (p65), Stat1, Tlr4, and Il6, in WAT (p < 0.05). Using microbial 16S RNA sequencing, it is demonstrated that the relative abundance of the Lactococcus, Parasutterella, and Turicibacter genera are increased in the HFC group versus HFD. CONCLUSIONS: Curcumin exerts protective metabolic effects in dietary obesity, in part through downregulation of adipose tissue inflammation, which may be mediated by alterations in composition of gut microbiota, and metabolism of curcumin into curcumin-O-glucuronide.

Functional significance of gain-of-function H19 lncRNA in skeletal muscle differentiation and anti-obesity effects.

BACKGROUND: Exercise training is well established as the most effective way to enhance muscle performance and muscle building. The composition of skeletal muscle fiber type affects systemic energy expenditures, and perturbations in metabolic homeostasis contribute to the onset of obesity and other metabolic dysfunctions. Long noncoding RNAs (lncRNAs) have been demonstrated to play critical roles in diverse cellular processes and diseases, including human cancers; however, the functional importance of lncRNAs in muscle performance, energy balance, and obesity remains elusive. We previously reported that the lncRNA H19 regulates the poly-ubiquitination and protein stability of dystrophin (DMD) in muscular dystrophy. METHODS: Here, we identified mouse/human H19-interacting proteins using mouse/human skeletal muscle tissues and liquid chromatography-mass spectrometry (LC-MS). Human induced pluripotent stem-derived skeletal muscle cells (iPSC-SkMC) from a healthy donor and Becker Muscular Dystrophy (BMD) patients were utilized to study DMD post-translational modifications and associated proteins. We identified a gain-of-function (GOF) mutant of H19 and characterized the effects on myoblast differentiation and fusion to myotubes using iPSCs. We then conjugated H19 RNA gain-of-function oligonucleotides (Rgof) with the skeletal muscle enrichment peptide agrin (referred to as AGR-H19-Rgof) and evaluated AGR-H19-Rgof's effects on skeletal muscle performance using wild-type (WT) C57BL/6 J mice and its anti-obesity effects using high-fat diet (HFD)- and leptin deficiency-induced obese mouse models. RESULTS: We demonstrated that both human and mouse H19 associated with DMD and that the H19 GOF exhibited enhanced interaction with DMD compared to WT H19. DMD was found to associate with serine/threonine-protein kinase MRCK alpha (MRCKα) and α-synuclein (SNCA) in iPSC-SkMC derived from BMD patients. Inhibition of MRCKα and SNCA-mediated phosphorylation of DMD antagonized the interaction between H19 and DMD. These signaling events led to improved skeletal muscle cell differentiation and myotube fusion. The administration of AGR-H19-Rgof improved the muscle mass, muscle performance, and base metabolic rate of WT mice. Furthermore, mice treated with AGR-H19-Rgof exhibited resistance to HFD- or leptin deficiency-induced obesity. CONCLUSIONS: Our study suggested the functional importance of the H19 GOF mutant in enhancing muscle performance and anti-obesity effects.

Co-delivery of novel bispecific and trispecific engagers by an amplicon vector augments the therapeutic effect of an HSV-based oncolytic virotherapy.

BACKGROUND: Although oncolytic virotherapy has shown substantial promises as a new treatment modality for many malignancies, further improvement on its therapeutic efficacy will likely bring more clinical benefits. One plausible way of enhancing the therapeutic effect of virotherapy is to enable it with the ability to concurrently engage the infiltrating immune cells to provide additional antitumor mechanisms. Here, we report the construction and evaluation of two novel chimeric molecules (bispecific chimeric engager proteins, BiCEP and trispecific chimeric engager protein, TriCEP) that can engage both natural killer (NK) and T cells with tumor cells for enhanced antitumor activities. METHODS: BiCEP was constructed by linking orthopoxvirus major histocompatibility complex class I-like protein, which can selectively bind to NKG2D with a high affinity to a mutant form of epidermal growth factor (EGF) that can strongly bind to EGF receptor. TriCEP is similarly constructed except that it also contains a modified form of interleukin-2 that can only function as a tethered form. As NKG2D is expressed on both NK and CD8+ T cells, both of which can thus be engaged by BiCEP and TriCEP. RESULTS: Both BiCEP and TriCEP showed the ability to engage NK and T cells to kill tumor cells in vitro. Coadministration of BiCEP and TriCEP with an oncolytic herpes simplex virus enhanced the overall antitumor effect. Furthermore, single-cell RNA sequencing analysis revealed that TriCEP not only engaged NK and T cells to kill tumor cells, it also promotes the infiltration and activation of these important immune cells. CONCLUSIONS: These novel chimeric molecules exploit the ability of the oncolytic virotherapy in altering the tumor microenvironment with increased infiltration of important immune cells such as NK and T cells for cancer immunotherapy. The ability of BiCEP and TriCEP to engage both NK and T cells makes them an ideal choice for arming an oncolytic virotherapy.

Functional and structural characterization of Hyp730, a highly conserved and dormancy-specific hypothetical membrane protein.

Membrane proteins represent major drug targets, and the ability to determine their functions, structures, and conformational changes will significantly advance mechanistic approaches to both biotechnology and bioremediation, as well as the fight against pathogenic bacteria. A pertinent example is Mycobacterium tuberculosis (H37Rv), which contains ~4000 protein-coding genes, with almost a thousand having been categorized as 'membrane protein', and a few of which (~1%) have been functionally characterized and structurally modeled. However, the functions and structures of most membrane proteins that are sparsely, or only transiently, expressed, but essential in small phenotypic subpopulations or under stress conditions such as persistence or dormancy, remain unknown. Our deep quantitative proteomics profiles revealed that the hypothetical membrane protein 730 (Hyp730) WP_010079730 (protein ID Mlut_RS11895) from M. luteus is upregulated in dormancy despite a ~5-fold reduction in overall protein diversity. Its H37Rv paralog, Rv1234, showed a similar proteomic signature, but the function of Hyp730-like proteins has never been characterized. Here, we present an extensive proteomic and transcriptomic analysis of Hyp730 and have also characterized its in vitro recombinant expression, purification, refolding, and essentiality as well as its tertiary fold. Our biophysical studies, circular dichroism, and tryptophan fluorescence are in immediate agreement with in-depth in silico 3D-structure prediction, suggesting that Hyp730 is a double-pass membrane-spanning protein. Ablation of Hyp730-expression did not alter M. luteus growth, indicating that Hyp730 is not essential. Structural homology comparisons showed that Hyp730 is highly conserved and non-redundant in G+C rich Actinobacteria and might be involved, under stress conditions, in an energy-saving role in respiration during dormancy.

Diversity of peripheral blood human NK cells identified by single-cell RNA sequencing.

Human natural killer (NK) cells in peripheral blood perform many functions, and classification of specific subsets has been a longstanding goal. We report single-cell RNA sequencing of NK cells, comparing gene expression in unstimulated and interleukin (IL)-2-activated cells from healthy cytomegalovirus (CMV)-negative donors. Three NK cell subsets resembled well-described populations; CD56brightCD16-, CD56dimCD16+CD57-, and CD56dimCD16+CD57+. CD56dimCD16+CD57- cells subdivided to include a population with higher chemokine mRNA and increased frequency of killer-cell immunoglobulin-like receptor expression. Three novel human blood NK cell populations were identified: a population of type I interferon-responding NK cells that were CD56neg; a population exhibiting a cytokine-induced memory-like phenotype, including increased granzyme B mRNA in response to IL-2; and finally, a small population, with low ribosomal expression, downregulation of oxidative phosphorylation, and high levels of immediate early response genes indicative of cellular activation. Analysis of CMV+ donors established that CMV altered the proportion of NK cells in each subset, especially an increase in adaptive NK cells, as well as gene regulation within each subset. Together, these data establish an unexpected diversity in blood NK cells and provide a new framework for analyzing NK cell responses in health and disease.

Wnt signaling regulates neural plate patterning in distinct temporal phases with dynamic transcriptional outputs.

The process that partitions the nascent vertebrate central nervous system into forebrain, midbrain, hindbrain, and spinal cord after neural induction is of fundamental interest in developmental biology, and is known to be dependent on Wnt/ß-catenin signaling at multiple steps. Neural induction specifies neural ectoderm with forebrain character that is subsequently posteriorized by graded Wnt signaling: embryological and mutant analyses have shown that progressively higher levels of Wnt signaling induce progressively more posterior fates. However, the mechanistic link between Wnt signaling and the molecular subdivision of the neural ectoderm into distinct domains in the anteroposterior (AP) axis is still not clear. To better understand how Wnt mediates neural AP patterning, we performed a temporal dissection of neural patterning in response to manipulations of Wnt signaling in zebrafish. We show that Wnt-mediated neural patterning in zebrafish can be divided into three phases: (I) a primary AP patterning phase, which occurs during gastrulation, (II) a mes/r1 (mesencephalon-rhombomere 1) specification and refinement phase, which occurs immediately after gastrulation, and (III) a midbrain-hindbrain boundary (MHB) morphogenesis phase, which occurs during segmentation stages. A major outcome of these Wnt signaling phases is the specification of the major compartment divisions of the developing brain: first the MHB, then the diencephalic-mesencephalic boundary (DMB). The specification of these lineage divisions depends upon the dynamic changes of gene transcription in response to Wnt signaling, which we show primarily involves transcriptional repression or indirect activation. We show that otx2b is directly repressed by Wnt signaling during primary AP patterning, but becomes resistant to Wnt-mediated repression during late gastrulation. Also during late gastrulation, Wnt signaling becomes both necessary and sufficient for expression of wnt8b, en2a, and her5 in mes/r1. We suggest that the change in otx2b response to Wnt regulation enables a transition to the mes/r1 phase of Wnt-mediated patterning, as it ensures that Wnts expressed in the midbrain and MHB do not suppress midbrain identity, and consequently reinforce formation of the DMB. These findings integrate important temporal elements into our spatial understanding of Wnt-mediated neural patterning and may serve as an important basis for a better understanding of neural patterning defects that have implications in human health.

Conversion of human cardiac progenitor cells into cardiac pacemaker-like cells.

We used a screening strategy to test for reprogramming factors for the conversion of human cardiac progenitor cells (CPCs) into Pacemaker-like cells. Human transcription factors SHOX2, TBX3, TBX5, TBX18, and the channel protein HCN2, were transiently induced as single factors and in trio combinations into CPCs, first transduced with the connexin 30.2 (CX30.2) mCherry reporter. Following screens for reporter CX30.2 mCherry gene activation and FACS enrichment, we observed the definitive expression of many pacemaker specific genes; including, CX30.2, KCNN4, HCN4, HCN3, HCN1, and SCN3b. These findings suggest that the SHOX2, HCN2, and TBX5 (SHT5) combination of transcription factors is a much better candidate in driving the CPCs into Pacemaker-like cells than other combinations and single transcription factors. Additionally, single-cell RNA sequencing of SHT5 mCherry+ cells revealed cellular enrichment of pacemaker specific genes including TBX3, KCNN4, CX30.2, and BMP2, as well as pacemaker specific potassium and calcium channels (KCND2, KCNK2, and CACNB1). In addition, similar to human and mouse sinoatrial node (SAN) studies, we also observed the down-regulation of NKX2.5. Patch-clamp recordings of the converted Pacemaker-like cells exhibited HCN currents demonstrated the functional characteristic of pacemaker cells. These studies will facilitate the development of an optimal Pacemaker-like cell-based therapy within failing hearts through the recovery of SAN dysfunction.

A Universal Stress Protein That Controls Bacterial Stress Survival in Micrococcus luteus.

Bacteria have remarkable mechanisms to survive severe external stresses, and one of the most enigmatic is the nonreplicative persistent (NRP) state. Practically, NRP bacteria are difficult to treat, and so inhibiting the proteins underlying this survival state may render such bacteria more susceptible to external stresses, including antibiotics. Unfortunately, we know little about the proteins and mechanisms conferring survival through the NRP state. Here, we report that a universal stress protein (Usp) is a primary regulator of bacterial survival through the NRP state in Micrococcus luteus NCTC 2665, a biosafety level 1 (BSL1) mycobacterial relative. Usps are widely conserved, and bacteria, including Mycobacterium tuberculosis, Mycobacterium smegmatis, and Escherichia coli, have multiple paralogs with overlapping functions that have obscured their functional roles. A kanamycin resistance cassette inserted into the M. luteus universal stress protein A 616 gene (ΔuspA616::kanM. luteus) ablates the UspA616 protein and drastically impairs M. luteus survival under even short-term starvation (survival, 83% wild type versus 32% ΔuspA616::kanM. luteus) and hypoxia (survival, 96% wild type versus 48% ΔuspA616::kanM. luteus). We observed no detrimental UspA616 knockout phenotype in logarithmic growth. Proteomics demonstrated statistically significant log-phase upregulation of glyoxylate pathway enzymes isocitrate lyase and malate synthase in ΔuspA616::kanM. luteus We note that these enzymes and the M. tuberculosis UspA616 homolog (Rv2623) are important in M. tuberculosis virulence and chronic infection, suggesting that Usps are important stress proteins across diverse bacterial species. We propose that UspA616 is a metabolic switch that controls survival by regulating the glyoxylate shunt.IMPORTANCE Bacteria tolerate severe external stresses, including antibiotics, through a nonreplicative persistent (NRP) survival state, yet the proteins regulating this survival state are largely unknown. We show a specific universal stress protein (UspA616) controls the NRP state in Micrococcus luteus Usps are widely conserved across bacteria, but their biological function(s) has remained elusive. UspA616 inactivation renders M. luteus susceptible to stress: bacteria die instead of adapting through the NRP state. UspA616 regulates malate synthase and isocitrate lyase, glyoxylate pathway enzymes important for chronic Mycobacterium tuberculosis infection. These data show that UspA616 regulates NRP stress survival in M. luteus and suggest a function for homologous proteins in other bacteria. Importantly, inhibitors of UspA616 and homologs may render NRP bacteria more susceptible to stresses, including current antibiotics.

Single-Cell RNA-Sequencing Identifies Activation of TP53 and STAT1 Pathways in Human T Lymphocyte Subpopulations in Response to Ex Vivo Radiation Exposure.

Detrimental health consequences from exposure to space radiation are a major concern for long-duration human exploration missions to the Moon or Mars. Cellular responses to radiation are expected to be heterogeneous for space radiation exposure, where only high-energy protons and other particles traverse a fraction of the cells. Therefore, assessing DNA damage and DNA damage response in individual cells is crucial in understanding the mechanisms by which cells respond to different particle types and energies in space. In this project, we identified a cell-specific signature for radiation response by using single-cell transcriptomics of human lymphocyte subpopulations. We investigated gene expression in individual human T lymphocytes 3 h after ex vivo exposure to 2-Gy gamma rays while using the single-cell sequencing technique (10X Genomics). In the process, RNA was isolated from ~700 irradiated and ~700 non-irradiated control cells, and then sequenced with ~50 k reads/cell. RNA in each of the cells was distinctively barcoded prior to extraction to allow for quantification for individual cells. Principal component and clustering analysis of the unique molecular identifier (UMI) counts classified the cells into three groups or sub-types, which correspond to CD4+, naïve, and CD8+/NK cells. Gene expression changes after radiation exposure were evaluated using negative binomial regression. On average, BBC3, PCNA, and other TP53 related genes that are known to respond to radiation in human T cells showed increased activation. While most of the TP53 responsive genes were upregulated in all groups of cells, the expressions of IRF1, STAT1, and BATF were only upregulated in the CD4+ and naïve groups, but were unchanged in the CD8+/NK group, which suggests that the interferon-gamma pathway does not respond to radiation in CD8+/NK cells. Thus, single-cell RNA sequencing technique was useful for simultaneously identifying the expression of a set of genes in individual cells and T lymphocyte subpopulation after gamma radiation exposure. The degree of dependence of UMI counts between pairs of upregulated genes was also evaluated to construct a similarity matrix for cluster analysis. The cluster analysis identified a group of TP53-responsive genes and a group of genes that are involved in the interferon gamma pathway, which demonstrate the potential of this method for identifying previously unknown groups of genes with similar expression patterns.

Development of a rapid UPLC-MS/MS determination of urine sulfocysteine for diagnosis of sulfocysteinuria and molybdenum co-factor deficiencies.

AIM: Molybdenum co-factor deficiencies and isolated sulfite oxidase deficiency are rare autosomal recessively inherited diseases characterized by severe psychomotor impairment, intractable seizures, dislocated lens and dysmorphic facial features. The biochemical diagnosis of these diseases requires the determination of urine sulfocysteine. MATERIALS & METHODS: Urine sulfocysteine was quantified by an ultra-high performance liquid chromatography-MS/MS assay. The method was validated for linearity, accuracy, precision, recovery and stability. RESULTS & CONCLUSION: Total imprecision of accuracy was less than 6%. Intra-assay and inter-assay precisions were less than 5%. The recovery was higher than 98%. The method is inexpensive, fast, accurate and has been successfully used for identifying five molybdenum co-factor deficient and six sulfite oxidase deficient patients since deployed.

Simultaneous determination of plasma total homocysteine and methionine by liquid chromatography-tandem mass spectrometry.

The sulfur-containing amino acid homocysteine is a cardiac risk factor and a biomarker for several inborn errors of metabolism in methionine synthesis. A simple LC-MS/MS method was developed and validated for determination of homocysteine and methionine in human plasma. Rapid separation was achieved using a reverse phase liquid chromatography. Mass spectrometry identification was performed in positive electrospray ionization mode for homocysteine and methionine. Accuracy, precision, linearity, recovery and sample stability were evaluated in the method validation. The test is applied in diagnosis of homocystinuria and monitoring total homocysteine levels. Moreover, simultaneous measurement of methionine helps in the differentiation of homocystinuria and some cobalamin disorders (such as cblC and cblD defects) without additional amino acid testing. Lastly, this assay is sensitive to detect reduced total homocysteine levels that are possibly seen in sulfocysteinuria and molybdenum cofactor deficiencies.