Analysis of RBP expression and binding sites identifies PTBP1 as a regulator of CD19 expression in B-ALL

Despite massive improvements in the treatment of B-ALL through CART-19 immunotherapy, a large number of patients suffer a relapse due to loss of the targeted epitope. Mutations in the CD19 locus and aberrant splicing events are known to account for the absence of surface antigen. However, early mole...

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Main Authors: Nicole Ziegler, Mariela Cortés-López, Francesca Alt, Maximilian Sprang, Arsenij Ustjanzew, Nadine Lehmann, Khalifa El Malki, Arthur Wingerter, Alexandra Russo, Olaf Beck, Sebastian Attig, Lea Roth, Julian König, Claudia Paret, Jörg Faber
Format: Article
Language:English
Published: Taylor & Francis Group 2023-12-01
Series:OncoImmunology
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Online Access:https://www.tandfonline.com/doi/10.1080/2162402X.2023.2184143
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author Nicole Ziegler
Mariela Cortés-López
Francesca Alt
Maximilian Sprang
Arsenij Ustjanzew
Nadine Lehmann
Khalifa El Malki
Arthur Wingerter
Alexandra Russo
Olaf Beck
Sebastian Attig
Lea Roth
Julian König
Claudia Paret
Jörg Faber
author_facet Nicole Ziegler
Mariela Cortés-López
Francesca Alt
Maximilian Sprang
Arsenij Ustjanzew
Nadine Lehmann
Khalifa El Malki
Arthur Wingerter
Alexandra Russo
Olaf Beck
Sebastian Attig
Lea Roth
Julian König
Claudia Paret
Jörg Faber
author_sort Nicole Ziegler
collection DOAJ
description Despite massive improvements in the treatment of B-ALL through CART-19 immunotherapy, a large number of patients suffer a relapse due to loss of the targeted epitope. Mutations in the CD19 locus and aberrant splicing events are known to account for the absence of surface antigen. However, early molecular determinants suggesting therapy resistance as well as the time point when first signs of epitope loss appear to be detectable are not enlightened so far. By deep sequencing of the CD19 locus, we identified a blast-specific 2-nucleotide deletion in intron 2 that exists in 35% of B-ALL samples at initial diagnosis. This deletion overlaps with the binding site of RNA binding proteins (RBPs) including PTBP1 and might thereby affect CD19 splicing. Moreover, we could identify a number of other RBPs that are predicted to bind to the CD19 locus being deregulated in leukemic blasts, including NONO. Their expression is highly heterogeneous across B-ALL molecular subtypes as shown by analyzing 706 B-ALL samples accessed via the St. Jude Cloud. Mechanistically, we show that downregulation of PTBP1, but not of NONO, in 697 cells reduces CD19 total protein by increasing intron 2 retention. Isoform analysis in patient samples revealed that blasts, at diagnosis, express increased amounts of CD19 intron 2 retention compared to normal B cells. Our data suggest that loss of RBP functionality by mutations altering their binding motifs or by deregulated expression might harbor the potential for the disease-associated accumulation of therapy-resistant CD19 isoforms.
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spelling doaj-art-2b706a5b32624427bf13e1bdcb10eeb52025-08-20T02:01:29ZengTaylor & Francis GroupOncoImmunology2162-402X2023-12-0112110.1080/2162402X.2023.2184143Analysis of RBP expression and binding sites identifies PTBP1 as a regulator of CD19 expression in B-ALLNicole Ziegler0Mariela Cortés-López1Francesca Alt2Maximilian Sprang3Arsenij Ustjanzew4Nadine Lehmann5Khalifa El Malki6Arthur Wingerter7Alexandra Russo8Olaf Beck9Sebastian Attig10Lea Roth11Julian König12Claudia Paret13Jörg Faber14Center for Pediatric and Adolescent Medicine, Department of Pediatric Hematology/Oncology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, GermanyInstitute of Molecular Biology (IMB), Mainz, GermanyCenter for Pediatric and Adolescent Medicine, Department of Pediatric Hematology/Oncology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, GermanyFaculty of Biology, Johannes Gutenberg University Mainz, Biozentrum I, Mainz, GermanyInstitute of Medical Biostatistics, Epidemiology and Informatics (IMBEI), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, GermanyCenter for Pediatric and Adolescent Medicine, Department of Pediatric Hematology/Oncology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, GermanyCenter for Pediatric and Adolescent Medicine, Department of Pediatric Hematology/Oncology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, GermanyCenter for Pediatric and Adolescent Medicine, Department of Pediatric Hematology/Oncology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, GermanyCenter for Pediatric and Adolescent Medicine, Department of Pediatric Hematology/Oncology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, GermanyCenter for Pediatric and Adolescent Medicine, Department of Pediatric Hematology/Oncology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, GermanyDepartment of Translational Oncology and Immunology at the Institute of Immunology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, GermanyCenter for Pediatric and Adolescent Medicine, Department of Pediatric Hematology/Oncology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, GermanyInstitute of Molecular Biology (IMB), Mainz, GermanyCenter for Pediatric and Adolescent Medicine, Department of Pediatric Hematology/Oncology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, GermanyCenter for Pediatric and Adolescent Medicine, Department of Pediatric Hematology/Oncology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, GermanyDespite massive improvements in the treatment of B-ALL through CART-19 immunotherapy, a large number of patients suffer a relapse due to loss of the targeted epitope. Mutations in the CD19 locus and aberrant splicing events are known to account for the absence of surface antigen. However, early molecular determinants suggesting therapy resistance as well as the time point when first signs of epitope loss appear to be detectable are not enlightened so far. By deep sequencing of the CD19 locus, we identified a blast-specific 2-nucleotide deletion in intron 2 that exists in 35% of B-ALL samples at initial diagnosis. This deletion overlaps with the binding site of RNA binding proteins (RBPs) including PTBP1 and might thereby affect CD19 splicing. Moreover, we could identify a number of other RBPs that are predicted to bind to the CD19 locus being deregulated in leukemic blasts, including NONO. Their expression is highly heterogeneous across B-ALL molecular subtypes as shown by analyzing 706 B-ALL samples accessed via the St. Jude Cloud. Mechanistically, we show that downregulation of PTBP1, but not of NONO, in 697 cells reduces CD19 total protein by increasing intron 2 retention. Isoform analysis in patient samples revealed that blasts, at diagnosis, express increased amounts of CD19 intron 2 retention compared to normal B cells. Our data suggest that loss of RBP functionality by mutations altering their binding motifs or by deregulated expression might harbor the potential for the disease-associated accumulation of therapy-resistant CD19 isoforms.https://www.tandfonline.com/doi/10.1080/2162402X.2023.2184143CD19B-ALLsplicingisoformsCART19 therapyRBP
spellingShingle Nicole Ziegler
Mariela Cortés-López
Francesca Alt
Maximilian Sprang
Arsenij Ustjanzew
Nadine Lehmann
Khalifa El Malki
Arthur Wingerter
Alexandra Russo
Olaf Beck
Sebastian Attig
Lea Roth
Julian König
Claudia Paret
Jörg Faber
Analysis of RBP expression and binding sites identifies PTBP1 as a regulator of CD19 expression in B-ALL
OncoImmunology
CD19
B-ALL
splicing
isoforms
CART19 therapy
RBP
title Analysis of RBP expression and binding sites identifies PTBP1 as a regulator of CD19 expression in B-ALL
title_full Analysis of RBP expression and binding sites identifies PTBP1 as a regulator of CD19 expression in B-ALL
title_fullStr Analysis of RBP expression and binding sites identifies PTBP1 as a regulator of CD19 expression in B-ALL
title_full_unstemmed Analysis of RBP expression and binding sites identifies PTBP1 as a regulator of CD19 expression in B-ALL
title_short Analysis of RBP expression and binding sites identifies PTBP1 as a regulator of CD19 expression in B-ALL
title_sort analysis of rbp expression and binding sites identifies ptbp1 as a regulator of cd19 expression in b all
topic CD19
B-ALL
splicing
isoforms
CART19 therapy
RBP
url https://www.tandfonline.com/doi/10.1080/2162402X.2023.2184143
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