Aberrant Splicing in Lymphoid Malignancies and its Implications for Immunotherapy

CD19 is expressed on the surface of most B-cell neoplasms, including acute lymphoblastic leukemias (B-ALL). There it can be targeted by a variety of effective immunotherapeutic agents, such as CART-19. However, relapses with epitope loss occur in up to 20% of B-ALL treated with CD19-directed immunotherapy.

Using whole exome and RNA sequencing and copy number alteration analysis, we detected acquired hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some - but not all - relapse samples. We also identified two alternatively spliced CD19 mRNA species: one lacking exons 5-6 (Δex5-6), which encode the transmembrane domain, and another lacking exon 2 (Δex2), compromising surface localization of CD19. Pull-down and siRNA experiments identified SRSF3 as a key splicing factor involved in exon 2 retention and differentially expressed in primary versus relapsed B-ALL.

Using CRISPR/Cas9-mediated gene editing, we demonstrated that exon 2 skipping can effectively bypass deleterious exon 2 mutations in B-ALL cells and yield truncated CD19 protein variants, which fail to trigger killing by CART-19 but partly rescue defects in cell proliferation and pre-BCR signaling associated with CD19 loss. Thus, there exists a novel mechanism of resistance to immunotherapy, based on a combination of deleterious mutations and selection for alternatively spliced RNA isoforms.

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