Abstract 2445: Alternative splicing dictates receptor tyrosine kinase fates in glioblastoma

Abstract

Abstract Receptor tyrosine kinase (RTK) genes are frequently amplified and/or overexpressed in glioblastoma (GBM). RTK signaling can undergo two fates - attenuation via lysosomal degradation or prolonged signaling via recycling. Annexin A7 (ANXA7) is a membrane binding protein belonging to the Annexin family, presumed to participate in the endocytic pathway. ANXA7 is alternatively spliced (AS) by PTBP1, a ribonucleoprotein, and expressed as two isoforms - unspliced Isoform 1 (I1), containing cassette exon 6 and spliced Isoform 2 (I2). We determined that I1 and I2 have divergent functional effects on RTK fates in GBM – I1 sorts RTKs for degradation thereby attenuating signaling, while I2 preferentially recycles RTKs and potentiates signaling. We hypothesize that I1, by virtue of its cassette exon, has the motifs necessary to target multiple RTKs for degradation thereby diminishing tumorigenic signaling in GBM.We generated multiple GBM cell lines that express endogenous I2 (I2 cells), or endogenous I2 + exogenous I1 (I1 cells). We evaluated the influence of I1 and/or I2 expression on RTK levels, activation and downstream events using western blot and co-immunoprecipitation assays. Receptor trafficking was observed using proximity ligation and immunofluorescence (IF) assays tagging different components of the endocytic pathway. To elucidate the motifs required for I1-mediated sorting, we used site-directed mutagenesis and created truncated I1 mutants lacking domains deemed necessary for degradation. We observed upregulated levels of several tumorigenic RTKs such as EGFR, MET, PDGFRα and EGFRvIII in the I2 cells. In I1 cells, however, we observed reduced levels and activation of all these RTKs along with diminished activation of downstream pathways. Immunostaining with endocytic markers revealed that RTKs were rapidly trafficked to the lysosomes in I1 cells indicative of degradation. Conversely, in I2 cells, RTKs sorted to the recycling endosomes perpetuating signaling. In our truncated I1 mutant models, we observed significantly higher levels of activated and total EGFR in a mutant with a 11 amino acid deletion in the cassette exon region. IF analysis showed that EGFR colocalized with recycling endosomes in this truncated mutant indicating that the absence of this domain impairs sorting to the lysosomes and re-routes the receptor to the recycling pathway causing sustained signaling. Dysregulated AS allows tumors to alter the transcriptome in favor of isoforms that drive tumor progression. Our results show that AS of ANXA7 can dictate the fate of RTK signaling by regulating the interaction of I1 and I2 with divergent endocytic processes in GBM. Domains encoded by the cassette exon, essential for I1-mediated RTK attenuation, are spliced out leading to unchecked signaling and tumor progression. Together, these results demonstrate how AS regulates protein interactions and signal transduction at a molecular level in GBM. Citation Format: Sindhu Nair, Rajani Rajbhandari, Centdrika Hurt, Markus Bredel. Alternative splicing dictates receptor tyrosine kinase fates in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2445.