Dual-trigger model of CD20 escape: NONO regulation and cryptic splicing induced by transcript overload in pediatric...

The B-cell-specific marker CD20 is expressed in various B-cell malignancies, including B-cell acute lymphoblastic leukemia (B-ALL) and serves as a key target for immunotherapies. Reduced or absent CD20 expression has been associated with diminished responses to anti-CD20 antibodies and CD20 directed CAR T-cells. Antigen loss may arise from alternative splicing or transcriptional downregulation of MS4A1, the gene coding for CD20, a processes influenced by RNA- and DNA-binding proteins. NONO, a non-POU domain-containing octamer-binding protein implicated in several cancers, regulates CD20 surface expression.

To explore factors associated with heterogeneous CD20 expression, we quantified MS4A1 transcript levels, profiled MS4A1 messenger RNA (mRNA) isoforms, and analyzed NONO mRNA in pediatric B-ALL samples. In addition, we used an in vitro CRISPR/Cas9 knockout model to assess the effects of NONO loss on MS4A1 transcript abundance, isoform distribution, and transcript stability. Plasmid-based overexpression of MS4A1 was used to examine its effect on splicing.

Loss of NONO was associated with increased MS4A1 transcript levels without detectable changes in isoform distribution or stability, and NONO mRNA expression was negatively associated with MS4A1 mRNA expression in CD20-positive blasts. At diagnosis, two MS4A1 mRNA isoforms were detected in CD20-positive blasts: The wild-type (WT-CD20) and a shorter variant (D393-CD20), a Δ4-6 multi-exon-skipped isoform that yields a truncated intracellular protein inaccessible to CD20-directed immunotherapies. Although WT-CD20 was the dominant splice isoform, the D393/WT-CD20 ratio correlated positively with overall MS4A1 transcript abundance. High WT-CD20 transcript abundance further biased splicing toward the D393-CD20 isoform, indicating involvement of cryptic splice sites and potential re-splicing events at the level of mature MS4A1 mRNA.

Together, these findings are consistent with a model in which NONO expression and transcript-level dynamics of MS4A1 are associated with CD20 heterogeneity in pediatric B-ALL. These observations may contribute to understanding variability in CD20 expression and antigen availability in pediatric B-ALL.