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Principal Investigator: Ryan Englander
Organization: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
Fiscal Year: 2023
Award: $52,694
Funding agency: National Cancer Institute
PROJECT SUMMARY/ABSTRACT
Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality in the United States. Immune
checkpoint inhibitors (ICIs) like anti-PD-1 have increased overall survival in NSCLC, but most patients still do
not respond to treatment. Cancer vaccines that target tumor-specific antigens, known as neoantigens, may
increase the efficacy of ICIs and other immunotherapies by expanding neoantigen-reactive CD8+ T cells that
can recognize and destroy tumor cells. Alternative splicing is a ubiquitous post-transcriptional regulatory process
that allows cells to produce different mRNA and protein sequences from the same gene. Alternative splicing is
broadly dysregulated in many cancer types including NSCLC and may generate novel peptide sequences absent
from normal tissue that can be recognized as neoantigens by CD8+ T cells. To identify alternative splicing-
derived neoantigens in NSCLC, we used long-read RNA sequencing to comprehensively map full-length mRNA
isoforms in NSCLC tumors and predict the proteins they encode with high accuracy. We found 145,914 predicted
peptides that were specific to tumors and shared by up to 70% of NSCLC patients. To identify which of these
peptides might be immunogenic, we used immunopeptidomics to directly sequence peptides bound to MHC
Class I in three NSCLC cell lines. We identified 21 peptides that are bound to MHC Class I on NSCLC cells and
are encoded by tumor-specific alternatively spliced mRNA isoforms. These splicing-derived peptides are
potentially shared neoantigens that might represent vaccine targets for NSCLC. Therefore, Aim 1 will test
whether any of these 21 splicing-derived peptides can be recognized by CD8+ T cells from NSCLC patients. We
will examine whether patient CD8+ T cells can proliferate, secrete cytokines like interferon-gamma, and lyse
target cells in response to these peptides. The experiments proposed in Aim 1 will provide crucial insight into the
frequency and immunogenicity of alternative splicing-derived neoantigens in NSCLC. Aim 2 will examine which
regulators of alternative splicing are driving production of these peptides. To this end, we will leverage publicly
available databases to identify splicing factors whose expression in tumors or target binding sites suggest an
association with the mRNA isoforms that code for the 21 splicing-derived peptides. We will use targeted genetic
approaches to study whether candidate splicing factors directly regulate peptide-coding isoform splicing in vitro.
This work will highlight mechanisms that can drive the production of tumor-specific splicing-derived peptides and
may reveal novel targets that can be exploited to enhance NSCLC immunogenicity. Altogether, these studies
may identify candidates for new immunotherapies, including personalized NSCLC cancer vaccines that can be
used to treat multiple patients who share expression of immunogenic splicing-derived neoantigens. This proposal
will provide me excellent training that will facilitate my career goals as a physician-scientist who leverages
advances in genomics and immunology to improve care for patients with cancer.
Terms: <Alternate Splicing><Alternative RNA Splicing><Alternative Splicing><Amino Acid Sequence><Antigen Targeting><Antigens><Antineoplastic Vaccine><Assay><Automobile Driving><Binding><Binding Sites><Bioassay><Biologic Assays><Biological Assay><Burkitt Herpesvirus><Burkitt Lymphoma Virus><CD8 Cell><CD8 T cells><CD8 lymphocyte><CD8+ T cell><CD8+ T-Lymphocyte><CD8-Positive Lymphocytes><CD8-Positive T-Lymphocytes><CMV><Cancer Cause><Cancer Etiology><Cancer Patient><Cancer Vaccines><Cancer cell line><Cancers><Cell Body><Cell surface><Cells><Cessation of life><Checkpoint inhibitor><Class I Genes><Co-culture><Cocultivation><Coculture><Coculture Techniques><Code><Coding System><Combining Site><Cytomegalovirus><Data><Data Bases><Databases><Death><Development><Disease><Disorder><EB virus><EBV><Epstein Barr Virus><Exons><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><Frequencies><GTEx><Genes><Genomics><Genotype-Tissue Expression Project><Goals><Grippe><HCMV><HHV-4><HHV4><Human><Human Herpesvirus 4><IFN-Gamma><IFN-g><IFN-γ><IFNG><IFNγ><Immune Interferon><Immune Precipitation><Immune checkpoint inhibitor><Immune mediated therapy><Immune response><Immunological response><Immunologically Directed Therapy><Immunology><Immunoprecipitation><Immunotherapy><In Vitro><Infectious Mononucleosis Virus><Influenza><Interferon Gamma><Interferon Type II><Intervening Sequences><Introns><Isoforms><Knock-out><Knockout><Lead><Length><MHC Class I><MHC Class I Genes><Malignant Cell><Malignant Neoplasms><Malignant Tumor><Maps><Measurement><Measures><Mediating><Messenger RNA><Methods><Modern Man><Molecular Interaction><NSCLC><NSCLC - Non-Small Cell Lung Cancer><Neoplasm Vaccines><Non-Small Cell Lung Cancer><Non-Small-Cell Lung Carcinoma><Nonsmall Cell Lung Carcinoma><Normal Tissue><Normal tissue morphology><PBMC><Pathway interactions><Patient Care><Patient Care Delivery><Patients><Pb element><Peptides><Peripheral Blood Mononuclear Cell><Physicians><Physiologic pulse><Pre-mRNA><Primary Neoplasm><Primary Protein Structure><Primary Tumor><Process><Production><Proliferating><Protein Isoforms><Proteins><Pulse><RNA Seq><RNA Splicing><RNA sequencing><RNA, Messenger, Precursors><RNA-Binding Proteins><RNAseq><Reactive Site><Research><Salivary Gland Viruses><Sampling><Scientist><Solvents><Sorting><Source><Splicing><Surface><T cell response><T-Cell Activation><T-Cells><T-Lymphocyte><T8 Cells><T8 Lymphocytes><TCGA><Techniques><Testing><The Cancer Genome Atlas><Training><Transcription Process><Translating><Tumor Antigens><Tumor Cell><Tumor Expansion><Tumor Vaccines><Tumor-Associated Antigen><United States><Vaccines><Viral><Work><aPD-1><aPD1><activate T cells><anti programmed cell death 1><anti-PD-1><anti-PD1><anti-programmed cell death protein 1><anti-tumor immune response><anti-tumor vaccine><antiPD-1><antiPD1><antitumor immune response><antitumor vaccine><bio-informatics pipeline><bioinformatics pipeline><cancer antigens><cancer cell><cancer type><candidate identification><career><cohort><crosslink><cytokine><cytomegalovirus group><data base><developmental><driving><experiment><experimental research><experimental study><experiments><flow cytophotometry><genetic approach><genetic strategy><heavy metal Pb><heavy metal lead><host response><immune check point inhibitor><immune system response><immune therapeutic approach><immune therapeutic interventions><immune therapeutic regimens><immune therapeutic strategy><immune therapy><immune-based therapies><immune-based treatments><immuno therapy><immunogen><immunogenic><immunogenicity><immunoresponse><improved><in silico><insight><lFN-Gamma><lung cancer cell><mRNA><mRNA Expression><mRNA Precursor><malignancy><mortality><neo-antigen><neo-epitopes><neoantigens><neoepitopes><neoplasm/cancer><neoplastic cell><novel><overexpress><overexpression><pathway><peptide aminoacid sequence><peptide sequence><posttranscriptional><protein aminoacid sequence><protein sequence><public data base><public database><publicly accessible data base><publicly accessible database><publicly available data base><publicly available database><response><thymus derived lymphocyte><transcriptome sequencing><transcriptomic sequencing><tumor><tumor specificity><tumor-specific antigen><vaccine for cancer><αPD-1><αPD1>