STEM-21. I1 EXCLUSIVE TYROSINE PHOSPHORYLATION IS REQUIRED FOR ANNEXIN A7 MEDIATED EGFR DEGRADATION IN GLIOMA STEM CELLS

Abstract Exon six of Annexin A7 (ANXA7) can be alternatively spliced to yield two distinct protein isoforms through its inclusion (I1) or exclusion (I2). Glioblastomas (GBMs) notably express aberrant receptor tyrosine kinase (RTK) activity, which are canonically endocytosed and degraded post-activation. However, this process is disrupted in GBM to promote receptor recycling, resulting in hyperactive RTK signaling. We have demonstrated that ANXA7 exon skipping supports the impaired endosomal trafficking of RTKs to favor recycling in established cells. Next, we sought to corroborate those findings further in a more clinically relevant GBM model. We identified clear ANXA7 isoform differences for the trafficking fates of epidermal growth factor receptor (EGFR) in glioma stem cells (GSCs) derived from patient tumors. Primary GSCs were made to overexpress ANXA7-I1 with lentivirus where it was found to be exceedingly toxic to virtually all patient tumors tested by the inability to garner I1 stably expressing cells. However, some patient GSCs were successfully transduced and then whole-cell lysates and surface only EGFR assessed by immunoblotting and FACS respectively. We identified I1 to promote EGFR degradation, portrayed by diminished total expression within GSCs. Furthermore, we observed a restoration of EGFR molecules to the cell surface 60 minutes after ligand stimulation in empty vector (EV) cells without I1 expression when previously down ~50% at the 30-minute time point. On the other hand, I1 expressing cells sustained diminished surface EGFR levels, suggesting their preference for receptor degradation versus recycling. We identified the phosphorylation of I1-exclusive tyrosine residues to be essential for this mechanism as exemplified through phosphomutants. Therefore, we implicated ANXA7 I1 as a potential therapeutic avenue for diminishing EGFR signaling in GSCs that may incite a novel vulnerability for this resistant tumor cell population by reducing their tumorigenicity, stemness capacity, or augmenting the efficacy of EGFR and other tyrosine kinase inhibitors.