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ABSTRACT
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.
Disclosure statement
The authors declare no competing financial interests.
This project has been funded by the NMFZ program of the University of Mainz and the Gilead funding program, the foundation “Kinderkrebsforschung Mainz”, the Walter Schulz foundation and the DFG.
Author contributions
NZ, MCL, MS, AU, CP were responsible for conducting and evaluating the experiments and preparing the figures. NZ, MS and AU performed statistical analysis. NZ and CP wrote the manuscript. LR provided technical assistance for sample preparation and flow cytometric analysis. NL assisted in the evaluation of the flow cytometry data. SA performed cell sorting. CP, JK and NZ designed the study. FA, KEM, AW, AR, OB provided the patient population. FA and KEM provided guidance for patients’ selection. FA and JF obtained the ethical approval for the study. CP and JF were responsible for project administration and supervision. CP, JF and JK acquired the funding. All authors have read and agreed to the published version of the manuscript.
Data Availability Statement
The authors confirm that the data supporting the findings of the present study are available within the article and its supplementary material. Raw data is available on request from the corresponding author [CP].
Following St. Jude Cloud datasets were used for RNAseq analysis: Pediatric Cancer Genome Project (PCGP): This study makes use of data generated by the St. Jude Children’s Research Hospital – Washington University Pediatric Cancer Genome Project and/or Childhood Solid Tumor Network.Citation53 Genomes for Kids (G4K): This study makes use of data generated by the St. Jude Children’s Research Hospital Genomes for Kids Study.Citation54 Real-Time Clinical Genomics (RTCG): This study makes use of data generated by St. Jude Children’s Research Hospital.Citation55 Pan-Acute Lymphoblastic Leukemia (PanALL): This study makes use of data generated by the Pan-Acute Lymphoblastic Leukemia Data Set of St. Jude Children’s Research Hospital.Citation56–59
Supplementary material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/2162402X.2023.2184143