To better understand and potentially predict CD19 loss-mediated resistance to CAR T cell therapy in patients with B-ALL, Cortés-López, Schulz, and Enculescu et al. investigated the effects of intronic and exonic mutations in the CD19 gene region encompassing exons 1-3 on splicing. They identified 200 single-point mutations that alter CD19 splicing and nearly 100 previously unknown splice isoforms, many of which emerged from cryptic (not observed in unmutated) splice sites and which likely code for non-functional CD19 molecules. They also identified 119 previously reported and novel RNA-binding proteins, including SF3B4 and PTBP1, that contribute to proper CD19 splicing.
Contributed by Lauren Hitchings
ABSTRACT: Following CART-19 immunotherapy for B-cell acute lymphoblastic leukaemia (B-ALL), many patients relapse due to loss of the cognate CD19 epitope. Since epitope loss can be caused by aberrant CD19 exon 2 processing, we herein investigate the regulatory code that controls CD19 splicing. We combine high-throughput mutagenesis with mathematical modelling to quantitatively disentangle the effects of all mutations in the region comprising CD19 exons 1-3. Thereupon, we identify ~200 single point mutations that alter CD19 splicing and thus could predispose B-ALL patients to developing CART-19 resistance. Furthermore, we report almost 100 previously unknown splice isoforms that emerge from cryptic splice sites and likely encode non-functional CD19 proteins. We further identify cis-regulatory elements and trans-acting RNA-binding proteins that control CD19 splicing (e.g., PTBP1 and SF3B4) and validate that loss of these factors leads to pervasive CD19 mis-splicing. Our dataset represents a comprehensive resource for identifying predictive biomarkers for CART-19 therapy.
Author Info: (1) Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany. (2) Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany. (3) Institute of M
Author Info: (1) Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany. (2) Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany. (3) Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany. (4) Department of Pediatric Hematology/Oncology, Center for Pediatric and Adolescent Medicine, University Medical Center of the Johannes Gutenberg University Mainz, 55131, Mainz, Germany. University Cancer Center (UCT), University Medical Center of the Johannes Gutenberg University Mainz, 55131, Mainz, Germany. German Cancer Consortium (DKTK), site Frankfurt/Mainz, Germany, German Cancer Research Center (DKFZ), 69120, Heidelberg, Germany. (5) Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany. (6) Department of Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA. Department of Biochemistry and Biophysics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA. (7) Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA. (8) Department of Systems Biology, Institute for Biomedical Genetics (IBMG), University of Stuttgart, Allmandring 30E, 70569, Stuttgart, Germany. (9) Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany. (10) Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany. (11) Department of Systems Biology, Institute for Biomedical Genetics (IBMG), University of Stuttgart, Allmandring 30E, 70569, Stuttgart, Germany. (12) Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany. (13) Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany. (14) Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA. Department of Biomedical and Health Informatics, Children's Hospital of Philadelphia, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA. (15) Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany. (16) Department of Pediatric Hematology/Oncology, Center for Pediatric and Adolescent Medicine, University Medical Center of the Johannes Gutenberg University Mainz, 55131, Mainz, Germany. University Cancer Center (UCT), University Medical Center of the Johannes Gutenberg University Mainz, 55131, Mainz, Germany. German Cancer Consortium (DKTK), site Frankfurt/Mainz, Germany, German Cancer Research Center (DKFZ), 69120, Heidelberg, Germany. (17) Department of Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA. (18) Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA. Department of Pathology & Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA. (19) Buchmann Institute for Molecular Life Sciences (BMLS), Max-von-Laue-Str. 15, 60438, Frankfurt, Germany. kathi.zarnack@bmls.de. Faculty Biological Sciences, Goethe University Frankfurt, Max-von-Laue-Str. 15, 60438, Frankfurt, Germany. kathi.zarnack@bmls.de. (20) Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany. legewie@ibmg.uni-stuttgart.de. Department of Systems Biology, Institute for Biomedical Genetics (IBMG), University of Stuttgart, Allmandring 30E, 70569, Stuttgart, Germany. legewie@ibmg.uni-stuttgart.de. Stuttgart Research Center for Systems Biology (SRCSB), University of Stuttgart, Stuttgart, Germany. legewie@ibmg.uni-stuttgart.de. (21) Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany. j.koenig@imb-mainz.de.
