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Principal Investigator: Paul Michael Armistead
Organization: UNIV OF NORTH CAROLINA CHAPEL HILL
Fiscal Year: 2020
Award: $450,736
Funding agency: National Cancer Institute
DESCRIPTION (provided by applicant): Chronic and acute leukemias are responsible for ~25,000 deaths annually in the US - a mortality rate that represents ~5% of all cancer related deaths. While leukemias are susceptible to antigen-specific T-cell mediated cytotoxicity, leukemia antigen (LA) discovery methods are complex and time consuming. Most LAs are derived from proteins that contain a somatic mutation or are aberrantly expressed or localized in the leukemia cell; however, another potential source of many LAs are antigenic peptides derived from leukemia specific protein splice isoforms. Our laboratory performed deep RNA-Sequencing of 8 acute myeloid leukemia (AML) samples, which did not contain spliceosome mutations, and identified >300,000 previously unannotated (i.e. novel) splice junctions. The central hypothesis of this project is that leukemia-specific splice junctions yield peptide epitopes that can be presented by class I HLA and targeted by CD8+ cytotoxic T-cells. Roughly 15% of AML and chronic lymphocytic leukemia (CLL) samples contain spliceosome mutations: primarily U2AF1 mutations in AML and SF3B1 mutations in CLL. In Aims 1 and 2, we will investigate the effect of these mutations on mRNA splicing, protein isoform generation and neo-antigen presentation in model cell lines modified to contain common U2AF1 and SF3B1 mutations (Aim 1) and then in human AML and CLL samples with or without a U2AF1 or SF3B1 mutation (Aim 2). In collaboration with Dr. Jan Prins, of the UNC Department of Computer Science, we will perform hybrid RNA-Sequencing (RNA-Seq) that combines short-read RNA-Seq data for splice junction discovery with long-read RNA-Seq to determine full mRNA isoform sequences. Following isoform sequencing we will test for protein translation of the isoform by probing for splice junction specific peptides using a prototype differential ion mobility spectrometer - mass spectrometer (DIMS-MS) that we have used with Dr. Gary Glish (UNC Department of Chemistry) on peptide discovery projects before. Aim 3 of this research proposal will apply these methods, as well as whole exome sequencing and comprehensive peptide epitope prediction performed by the lab of Dr. Catherine Wu, in a clinical protocol where we will genetically and computational predict LAs from AML or CLL patient samples, confirm their expression by DIMS-MS, and generate LA-specific T-cells from autologous patient-derived lymphocytes. At the completion of this project we will have characterized the effects of spliceosome mutations on mRNA splicing and its resultant effect on protein isoform production and epitope presentation. More significantly, we will have developed a methodology for personalized LA discovery, where we will identify and confirm patient-derived LA that are presented by the patient's HLA, and produce patient-derived LA-specific T-cells. The combined sequencing, bioinformatics and proteomics methods could be applied for the development of patient-specific T-cell therapeutics.
Terms: <AML - Acute Myeloid Leukemia><Acute Myeloblastic Leukemia><Acute Myelocytic Leukemia><Acute Myelogenous Leukemia><Acute leukemia><Adoptive Cell Transfers><American><Analytic Chemistry><Analytical Chemistry><Antigen Presentation><Antigenic Determinants><Antigens><Autologous><B-Cell CLL><B-Cell Chronic Lymphocytic Leukemia><B-Cell Chronic Lymphogenous Leukemia><B-Cell Chronic Lymphoid Leukemia><B-Cell Lymphocytic Leukemia><B-Lymphocytic Leukemia><Binding Determinants><Bio-Informatics><Bioinformatics><Biological><CD8 Cell><CD8 T cells><CD8 lymphocyte><CD8+ T cell><CD8+ T-Lymphocyte><CD8-Positive Lymphocytes><CD8-Positive T-Lymphocytes><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Cancers><Cas nuclease technology><Cell Body><Cell Line><Cell model><Cell-Mediated Cytolysis><Cell-Mediated Lympholysis><Cell-Mediated Lympholytic Cells><CellLine><Cells><Cellular Cytotoxicity><Cellular model><Cessation of life><Chemicals><Chemistry><Chronic B-Lymphocytic Leukemia><Chronic Lymphatic Leukemia><Chronic Lymphoblastic Leukemia><Chronic Lymphocytic Leukemia><Chronic Lymphogenous Leukemia><Clinical><Clinical Protocols><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Collaborations><Complex><Consumption><Cytolytic T-Cell><Cytotoxic T Cell><Cytotoxic T-Lymphocytes><DF/HCC><Dana-Farber Cancer Institute><Data><Death><Development><Epitopes><Funding><Gene Transcription><Generations><Genes><Genetic Alteration><Genetic Change><Genetic Transcription><Genetic defect><Genomics><Grant><Hematologic Cancer><Hematologic Malignancies><Hematologic Neoplasms><Hematological Malignancies><Hematological Neoplasms><Hematological Tumor><Hematopoietic Cancer><Human><Hybrids><Immune><Immune mediated therapy><Immune system><Immunes><Immunologically Directed Therapy><Immunotherapy><Isoforms><Laboratories><Length><Leukemic Cell><Lymphocyte><Lymphocyte Cytotoxicity><Lymphocytic><Lymphocytotoxicity><Malignant Hematologic Neoplasm><Malignant Neoplasms><Malignant Tumor><Manuscripts><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Measures><Messenger RNA><Methodology><Methods><Modern Man><Mutate><Mutation><North Carolina><Pathway interactions><Patients><Peptides><Pre-mRNA><Process><Production><Protein Isoforms><Protein Splicing><Proteins><Proteomics><Publishing><RNA Expression><RNA Seq><RNA Splicing><RNA sequencing><RNA, Messenger, Precursors><RNAseq><Recurrence><Recurrent><Research><Research Proposals><Role><Sampling><Somatic Mutation><Source><Spliceosomes><Splicing><Strains Cell Lines><Syndrome><System><T cell response><T-Cells><T-Lymphocyte><T8 Cells><T8 Lymphocytes><Techniques><Testing><Therapeutic><Therapy trial><Time><Transcription><Translations><Universities><VAC-TX><Vaccine Therapy><acute granulocytic leukemia><acute myeloid leukemia><adoptive cell therapy><adoptive cellular therapy><allergic/immunologic body system><allergic/immunologic organ system><antigen-specific T cells><bio-informatics pipeline><bioinformatics pipeline><cell mediated cytotoxicity><chronic leukemia><chronic lymphoid leukemia><comparative><computational chemistry><computer science><cultured cell line><cytotoxic CD8 T cells><cytotoxic CD8 T lymphocyte><developmental><differential expression><differentially expressed><exome sequencing><exome-seq><genome mutation><global gene expression><global transcription profile><immune therapeutic approach><immune therapeutic interventions><immune therapeutic regimens><immune therapeutic strategy><immune therapy><immune-based therapies><immune-based treatments><immuno therapy><immunogen><in vivo><ion mobility><killer T cell><leukemia><leukemic stem cell><lymph cell><mRNA><mRNA Precursor><malignancy><mass spectrometer><mortality><neo-antigen><neo-epitopes><neoantigens><neoepitopes><neoplasm/cancer><novel><pathway><premRNA><prototype><public health relevance><social role><stem cell population><tandem mass spectrometry><therapeutic target><therapeutic vaccination><thymus derived lymphocyte><transcriptional differences><transcriptome><transcriptome sequencing><transcriptomics>