Bioinformatics Methods for Prediction of Splice Variant Neoantigens

Tumor-specific peptide epitopes that are generated from mutated genes and presented on cell surface MHC molecules, known as neoantigens, are attractive targets for therapeutic vaccination given the lack of central tolerance and corresponding presence of endogenous T cells that recognize them. Currently most available neoantigen prediction methods focus on predicting neoantigens derived from missense mutations or indels. In acute myeloid leukemia (AML), there are markedly fewer mutations and predicted neoantigens in the cancer genome compared to other cancers, so it is less feasible to target neoantigens derived from missense mutations and indels in AML. However, mutations in spliceosomal genes and genome-wide aberrant splicing events are common in patients with AML. In work contributed to by our group, a small number of splice variant neoantigens have been found to exist in cancer. Herein, we report the development of robust method, NeoSplice, to predict splice variant neoantigens from massively parallel RNA sequencing (RNA-Seq) data. One of the computational challenges for predicting splice variant neoantigens is to infer the novel transcript isoforms derived from tumor-specific splicing events. We utilized a Burrows Wheeler Transform (BWT) based algorithm to identify tumor specific k-mers and used a splice graph to determine whether such a k-mer represents a tumor-specific splice junction in a coding region and its corresponding amino-acid sequence. A frame-shift relative to the normal can easily lead to a novel peptide sequence that may be an actionable neoantigen. Most current neoantigen calling algorithms primarily rely on epitope/MHC binding affinity predictions to rank and select for potential epitope targets. These algorithms do not predict for epitope immunogenicity using approaches modeled from tumor-specific antigen data. We developed an algorithm based on peptide-intrinsic biochemical features associated with neoantigen and minor histocompatibility mismatch antigen (mHA) immunogenicity and present a gradient–boosting algorithm for predicting tumor antigen immunogenicity. In addition, as part of PhD training in bioinformatics analysis to complement training in methods development, we performed comprehensive genomic and immune characterizations of bladder tumors and triple-negative breast cancer brain metastases to gain novel insight about biomarkers that can be used with potential immunotherapies.Doctor of Philosoph

HLAProfiler utilizes k-mer profiles to improve HLA calling accuracy for rare and common alleles in RNA-seq data

BACKGROUND: The human leukocyte antigen (HLA) system is a genomic region involved in regulating the human immune system by encoding cell membrane major histocompatibility complex (MHC) proteins that are responsible for self-recognition. Understanding the variation in this region provides important insights into autoimmune disorders, disease susceptibility, oncological immunotherapy, regenerative medicine, transplant rejection, and toxicogenomics. Traditional approaches to HLA typing are low throughput, target only a few genes, are labor intensive and costly, or require specialized protocols. RNA sequencing promises a relatively inexpensive, high-throughput solution for HLA calling across all genes, with the bonus of complete transcriptome information and widespread availability of historical data. Existing tools have been limited in their ability to accurately and comprehensively call HLA genes from RNA-seq data. RESULTS: We created HLAProfiler ( https://github.com/ExpressionAnalysis/HLAProfiler ), a k-mer profile-based method for HLA calling in RNA-seq data which can identify rare and common HLA alleles with > 99% accuracy at two-field precision in both biological and simulated data. For 68% of novel alleles not present in the reference database, HLAProfiler can correctly identify the two-field precision or exact coding sequence, a significant advance over existing algorithms. CONCLUSIONS: HLAProfiler allows for accurate HLA calls in RNA-seq data, reliably expanding the utility of these data in HLA-related research and enabling advances across a broad range of disciplines. Additionally, by using the observed data to identify potential novel alleles and update partial alleles, HLAProfiler will facilitate further improvements to the existing database of reference HLA alleles. HLAProfiler is available at https://expressionanalysis.github.io/HLAProfiler/

HiView: an integrative genome browser to leverage Hi-C results for the interpretation of GWAS variants

Abstract Background Genome-wide association studies (GWAS) have identified thousands of genetic variants associated with complex traits and diseases. However, most of them are located in the non-protein coding regions, and therefore it is challenging to hypothesize the functions of these non-coding GWAS variants. Recent large efforts such as the ENCODE and Roadmap Epigenomics projects have predicted a large number of regulatory elements. However, the target genes of these regulatory elements remain largely unknown. Chromatin conformation capture based technologies such as Hi-C can directly measure the chromatin interactions and have generated an increasingly comprehensive catalog of the interactome between the distal regulatory elements and their potential target genes. Leveraging such information revealed by Hi-C holds the promise of elucidating the functions of genetic variants in human diseases. Results In this work, we present HiView, the first integrative genome browser to leverage Hi-C results for the interpretation of GWAS variants. HiView is able to display Hi-C data and statistical evidence for chromatin interactions in genomic regions surrounding any given GWAS variant, enabling straightforward visualization and interpretation. Conclusions We believe that as the first GWAS variants-centered Hi-C genome browser, HiView is a useful tool guiding post-GWAS functional genomics studies. HiView is freely accessible at: http://www.unc.edu/~yunmli/HiView

Luteolin inhibits GPVI-mediated platelet activation, oxidative stress, and thrombosis

Introduction: Luteolin inhibits platelet activation and thrombus formation, but the mechanisms are unclear. This study investigated the effects of luteolin on GPVI-mediated platelet activation in vitro and explored the effect of luteolin on thrombosis, coagulation, and platelet production in vivo.Methods: Washed human platelets were used for aggregation, membrane protein expression, ATP, Ca2+, and LDH release, platelet adhesion/spreading, and clot retraction experiments. Washed human platelets were used to detect collagen and convulxin-induced reactive oxygen species production and endogenous antioxidant effects. C57BL/6 male mice were used for ferric chloride-induced mesenteric thrombosis, collagen-epinephrine induced acute pulmonary embolism, tail bleeding, coagulation function, and luteolin toxicity experiments. The interaction between luteolin and GPVI was analyzed using solid phase binding assay and surface plasmon resonance (SPR).Results: Luteolin inhibited collagen- and convulxin-mediated platelet aggregation, adhesion, and release. Luteolin inhibited collagen- and convulxin-induced platelet ROS production and increased platelet endogenous antioxidant capacity. Luteolin reduced convulxin-induced activation of ITAM and MAPK signaling molecules. Molecular docking simulation showed that luteolin forms hydrogen bonds with GPVI. The solid phase binding assay showed that luteolin inhibited the interaction between collagen and GPVI. Surface plasmon resonance showed that luteolin bonded GPVI. Luteolin inhibited integrin αIIbβ3-mediated platelet activation. Luteolin inhibited mesenteric artery thrombosis and collagen- adrenergic-induced pulmonary thrombosis in mice. Luteolin decreased oxidative stress in vivo. Luteolin did not affect coagulation, hemostasis, or platelet production in mice.Discussion: Luteolin may be an effective and safe antiplatelet agent target for GPVI. A new mechanism (decreased oxidative stress) for the anti-platelet activity of luteolin has been identified

Antigen-capturing nanoparticles improve the abscopal effect and cancer immunotherapy

Immunotherapy holds tremendous promise for improving cancer treatment1. Administering radiotherapy with immunotherapy has been shown to improve immune responses and can elicit an “abscopal effect”2. Unfortunately, response rates for this strategy remain low3. Herein, we report an improved cancer immunotherapy approach that utilizes antigen-capturing nanoparticles (AC-NPs). We engineered several AC-NPs formulations and demonstrated that the set of protein antigens captured by each AC-NP formulation is dependent upon NP surface properties. We showed that AC-NPs deliver tumor specific proteins to antigen-presenting cells and significantly improve the efficacy of αPD-1 treatment using the B16F10 melanoma model, generating up to 20% cure rate as compared to 0% without AC-NPs. Mechanistic studies revealed that AC-NPs induced an expansion of CD8+ cytotoxic T cells and increased both CD4+/Treg and CD8+/Treg ratios. Our work presents a novel strategy for improving cancer immunotherapy with nanotechnology

Comprehensive Analysis of the Immunogenomics of Triple-Negative Breast Cancer Brain Metastases From LCCC1419

BackgroundTriple negative breast cancer (TNBC) is an aggressive variant of breast cancer that lacks the expression of estrogen and progesterone receptors (ER and PR) and HER2. Nearly 50% of patients with advanced TNBC will develop brain metastases (BrM), commonly with progressive extracranial disease. Immunotherapy has shown promise in the treatment of advanced TNBC; however, the immune contexture of BrM remains largely unknown. We conducted a comprehensive analysis of TNBC BrM and matched primary tumors to characterize the genomic and immune landscape of TNBC BrM to inform the development of immunotherapy strategies in this aggressive disease.MethodsWhole-exome sequencing (WES) and RNA sequencing were conducted on formalin-fixed, paraffin-embedded samples of BrM and primary tumors of patients with clinical TNBC (n = 25, n = 9 matched pairs) from the LCCC1419 biobank at UNC—Chapel Hill. Matched blood was analyzed by DNA sequencing as a comparison for tumor WES for the identification of somatic variants. A comprehensive genomics assessment, including mutational and copy number alteration analyses, neoantigen prediction, and transcriptomic analysis of the tumor immune microenvironment were performed.ResultsPrimary and BrM tissues were confirmed as TNBC (23/25 primaries, 16/17 BrM) by immunohistochemistry and of the basal intrinsic subtype (13/15 primaries and 16/19 BrM) by PAM50. Compared to primary tumors, BrM demonstrated a higher tumor mutational burden. TP53 was the most frequently mutated gene and was altered in 50% of the samples. Neoantigen prediction showed elevated cancer testis antigen- and endogenous retrovirus-derived MHC class I-binding peptides in both primary tumors and BrM and predicted that single-nucleotide variant (SNV)-derived peptides were significantly higher in BrM. BrM demonstrated a reduced immune gene signature expression, although a signature associated with fibroblast-associated wound healing was elevated in BrM. Metrics of T and B cell receptor diversity were also reduced in BrM.ConclusionsBrM harbored higher mutational burden and SNV-derived neoantigen expression along with reduced immune gene signature expression relative to primary TNBC. Immune signatures correlated with improved survival, including T cell signatures. Further research will expand these findings to other breast cancer subtypes in the same biobank. Exploration of immunomodulatory approaches including vaccine applications and immune checkpoint inhibition to enhance anti-tumor immunity in TNBC BrM is warranted

Tyrosine Kinase ETK/BMX Is Up-Regulated in Bladder Cancer and Predicts Poor Prognosis in Patients with Cystectomy

Deregulation of the non-receptor tyrosine kinase ETK/BMX has been reported in several solid tumors. In this report, we demonstrated that ETK expression is progressively increased during bladder cancer progression. We found that down-regulation of ETK in bladder cancer cells attenuated STAT3 and AKT activity whereas exogenous overexpression of ETK had opposite effects, suggesting that deregulation of ETK may attribute to the elevated activity of STAT3 and AKT frequently detected in bladder cancer. The survival, migration and invasion of bladder cancer cells were significantly compromised when ETK expression was knocked down by a specific shRNA. In addition, we showed that ETK localizes to mitochondria in bladder cancer cells through interacting with Bcl-XL and regulating ROS production and drug sensitivity. Therefore, ETK may play an important role in regulating survival, migration and invasion by modulating multiple signaling pathways in bladder cancer cells. Immunohistochemistry analysis on tissue microarrays containing 619 human bladder tissue samples shows that ETK is significantly upregulated during bladder cancer development and progression and ETK expression level predicts the survival rate of patients with cystectomy. Taken together, our results suggest that ETK may potentially serve as a new drug target for bladder cancer treatment as well as a biomarker which could be used to identify patients with higher mortality risk, who may be benefited from therapeutics targeting ETK activity

A Novel Secure Routing Design Based on Physical Layer Security in Millimeter-Wave VANET

With the continuous development of millimeter-wave communication technology, new requirements such as ultra-reliability and higher data rates pose new challenges to the security issues of traditional cryptographic encryption in vehicular ad hoc networks (VANET). Physical layer security uses the characteristics of different wireless channels to protect the information security. In this paper, we propose a novel VANET routing mechanism that utilizes physical layer security to improve the secrecy performance, which is compatible with the millimeter-wave vehicular network. Specifically, we design a new secure routing selection factor, the utility function, that takes into account the effects of both secrecy rate and single-hop transmission distance to achieve the hop selection. In addition, we propose a novel routing mechanism and design a waiting mechanism based on the utility function. Compared with the traditional routing algorithms, the greedy perimeter stateless routing (GPSR) and Dijkstra simulation results illustrate that our design achieves superior performance in secrecy performance and dynamic adaptability

Asymmetric full mode-converting transmission of elastic waves

Asymmetric transmission in which wave energy propagates only in one direction attracts significant attention in various fields because of its rich physics and potential applications. In this work, we propose an elastic mode-converting metamaterial, which allows a full-power mode-converting transmission from longitudinal waves to transverse waves in the forward direction, while completely restricts the L wave transmission in the inverse direction. The metamaterial is designed by simply cutting two arrays of periodic silts on a matrix by exploring a straight design methodology, and thus very friendly for fabrication and application. Eigen-frequency analysis shows that the bilayer metamaterial exhibits two modes with significantly close natural frequencies around the working frequency, one for full-power mode-converting transmission, and the other for asymmetric transmission. Ultrasonic experiments are carried out to validate the proposed design. Our work offers a simple and efficient way for the realization of a complete one-way mode-converting transmission, and could be critically useful in designing diode-like meta-devices for novel wave manipulations

Antiplatelet effects of the CEACAM1-derived peptide QDTT

AbstractCarcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) restricts platelet activation via platelet collagen receptor GPVI/FcRγ-chain. In this study, screening against collagen-induced platelet aggregation was performed to identify functional CEACAM1 extracellular domain fragments. CEACAM1 fragments, including Ala-substituted peptides, were synthesized. Platelet assays were conducted on healthy donor samples for aggregation, cytotoxicity, adhesion, spreading, and secretion. Mice were used for tail bleeding and FeCl3-induced thrombosis experiments. Clot retraction was assessed using platelet-rich plasma. Extracellular segments of CEACAM1 and A1 domain-derived peptide QDTT were identified, while N, A2, and B domains showed no involvement. QDTT inhibited platelet aggregation. Ala substitution for essential amino acids (Asp139, Thr141, Tyr142, Trp144, and Trp145) in the QDTT sequence abrogated collagen-induced aggregation inhibition. QDTT also suppressed platelet secretion and “inside-out” GP IIb/IIIa activation by convulxin, along with inhibiting PI3K/Akt pathways. QDTT curtailed FeCl3-induced mesenteric thrombosis without significantly prolonging bleeding time, implying the potential of CEACAM1 A1 domain against platelet activation without raising bleeding risk, thus paving the way for novel antiplatelet drugs