title: Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy.
abstract: The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms.
  
  
  CONCLUSIONS
  CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.
authors.affiliation: Division of Cancer Pathobiology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.
authors.forename: Elena
authors.initials: E
authors.lastname: Sotillo
authors.affiliation: Division of Oncology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.
authors.forename: David M
authors.initials: DM
authors.lastname: Barrett
authors.affiliation: Division of Cancer Pathobiology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.
authors.forename: Kathryn L
authors.initials: KL
authors.lastname: Black
authors.affiliation: Division of Cancer Pathobiology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.
authors.forename: Asen
authors.initials: A
authors.lastname: Bagashev
authors.affiliation: Division of Oncology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.
authors.forename: Derek
authors.initials: D
authors.lastname: Oldridge
authors.affiliation: Division of Cancer Pathobiology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania. Immunology Graduate Group, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.
authors.forename: Glendon
authors.initials: G
authors.lastname: Wu
authors.affiliation: Division of Oncology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.
authors.forename: Robyn
authors.initials: R
authors.lastname: Sussman
authors.affiliation: Division of Cancer Pathobiology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania. Cell & Molecular Biology Graduate Group, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.
authors.forename: Claudia
authors.initials: C
authors.lastname: Lanauze
authors.affiliation: Center for Cellular Immunotherapies, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.
authors.forename: Marco
authors.initials: M
authors.lastname: Ruella
authors.affiliation: Department of Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania. Department of Biochemistry & Biophysics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Gazzara
authors.affiliation: Department of Biochemistry & Biophysics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.
authors.forename: Nicole M
authors.initials: NM
authors.lastname: Martinez
authors.affiliation: Division of Cancer Pathobiology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania. Cell & Molecular Biology Graduate Group, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.
authors.forename: Colleen T
authors.initials: CT
authors.lastname: Harrington
authors.affiliation: Division of Cancer Pathobiology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.
authors.forename: Elaine Y
authors.initials: EY
authors.lastname: Chung
authors.affiliation: Division of Oncology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.
authors.forename: Jessica
authors.initials: J
authors.lastname: Perazzelli
authors.affiliation: Division of Oncology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.
authors.forename: Ted J
authors.initials: TJ
authors.lastname: Hofmann
authors.affiliation: Division of Oncology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.
authors.forename: Shannon L
authors.initials: SL
authors.lastname: Maude
authors.affiliation: Division of Cancer Pathobiology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania. Division of Oncology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.
authors.forename: Pichai
authors.initials: P
authors.lastname: Raman
authors.affiliation: Department of Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Barrera
authors.affiliation: Center for Cellular Immunotherapies, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania. Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.
authors.forename: Saar
authors.initials: S
authors.lastname: Gill
authors.affiliation: Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.
authors.forename: Simon F
authors.initials: SF
authors.lastname: Lacey
authors.affiliation: Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.
authors.forename: Jan J
authors.initials: JJ
authors.lastname: Melenhorst
authors.affiliation: Department of Pathology & Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.
authors.forename: David
authors.initials: D
authors.lastname: Allman
authors.affiliation: Pediatric Oncology Branch, National Cancer Institute, Bethesda, Maryland.
authors.forename: Elad
authors.initials: E
authors.lastname: Jacoby
authors.affiliation: Pediatric Oncology Branch, National Cancer Institute, Bethesda, Maryland.
authors.forename: Terry
authors.initials: T
authors.lastname: Fry
authors.affiliation: Pediatric Oncology Branch, National Cancer Institute, Bethesda, Maryland.
authors.forename: Crystal
authors.initials: C
authors.lastname: Mackall
authors.affiliation: Department of Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.
authors.forename: Yoseph
authors.initials: Y
authors.lastname: Barash
authors.affiliation: Department of Biochemistry & Biophysics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.
authors.forename: Kristen W
authors.initials: KW
authors.lastname: Lynch
authors.affiliation: Division of Oncology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.
authors.forename: John M
authors.initials: JM
authors.lastname: Maris
authors.affiliation: Division of Oncology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.
authors.forename: Stephan A
authors.initials: SA
authors.lastname: Grupp
authors.affiliation: Division of Cancer Pathobiology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania. Immunology Graduate Group, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania. Cell & Molecular Biology Graduate Group, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania. Department of Pathology & Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania. andreit@mail.med.upenn.edu.
authors.forename: Andrei
authors.initials: A
authors.lastname: Thomas-Tikhonenko
mesh_terms: D017398:Alternative Splicing; D018941:Antigens, CD19 / Q000235:genetics* / Q000378:metabolism; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D000939:Epitopes / Q000276:immunology; D005091:Exons; D006801:Humans; D007167:Immunotherapy / Q000379:methods; D009154:Mutation; D015452:Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics / Q000276:immunology / Q000473:pathology / Q000628:therapy; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics; D016601:RNA-Binding Proteins / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D012008:Recurrence; D017422:Sequence Analysis, DNA; D000068103:Serine-Arginine Splicing Factors; D014158:Transcription, Genetic
chemical_list: D018941:Antigens, CD19; D000939:Epitopes; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; C491388:SRSF3 protein, human; D000068103:Serine-Arginine Splicing Factors