NRCAM variant defined by microexon skipping is a targetable surface proteoform in high-grade gliomas

To overcome the paucity of known tumor-specific surface antigens in pediatric high-grade glioma (pHGG), we contrasted splicing patterns in pHGGs and normal brain samples. Among alternative splicing events affecting extracellular protein domains, the most pervasive alteration was the skipping of ≤30 nucleotide-long microexons. Several of these skipped microexons mapped to L1-IgCAM family members, such as NRCAM. Bulk and single-nuclei short- and long-read RNA-seq revealed uniform skipping of NRCAM microexons 5 and 19 in virtually every pHGG sample. Importantly, the Δex5Δex19 (but not the full-length) NRCAM proteoform was essential for pHGG cell migration and invasion in vitro and tumor growth in vivo. We developed a monoclonal antibody selective for Δex5Δex19 NRCAM and demonstrated that “painting“ of pHGG cells with this antibody enables killing by T cells armed with an FcRI-based universal immune receptor. Thus, pHGG-specific NRCAM and possibly other L1-IgCAM proteoforms are promising and highly selective targets for adoptive immunotherapies. Our recently developed SnISOr-Seq approach allows for the accurate cataloging of full-length transcripts devoid of intronic sequences. We applied this approach to the pediatric high-grade glioma cell line 7316-3058. The short-read part of the workflow was used to determine expression level of select genes in individual cells. The long-read part of the workflow was used to determine whether microexons of interest are processed in a uniform or in a stochastic manner.