ANGI-13. LONG NON-CODING RNA<i>LINC03045</i> REGULATES GLIOBLASTOMA INVASION VIA<i>WASF3</i>

Abstract Glioblastoma (GBM) is an aggressive, heterogeneous tumor characterized by extensive infiltration of tumor cells within the surrounding brain parenchyma. Invasive tumor cells that evade surgical resection, chemoradiation and immune responses underlie GBM recurrence, necessitating the evaluation of factors that govern them. A recently completed unbiased lncRNA CRISPRi screen done by our lab identified LINC03045 as the top potential regulator of GBM invasion in Matrigel assays. Since enhanced LINC03045 expression was also found to be significantly associated with glioma tumor grade and patient survival (p&amp;lt;0.0001), here we characterized potential mechanisms by which it regulates GBM invasion. Mediators of lncRNA effect were identified via differential expression analysis in LINC03045 knockdown glioma cells and TCGA glioma datasets, and assessed for tumor invasion in 2D and 3D assays using knockdown and rescue experiments. Differential expression analysis of protein-coding genes associated with reduced LINC03045 expression in vitro and in GBM TCGA RNAseq data revealed high positive correlation with WASF3 (p=0.016), a regulator of JAK-STAT signaling and cytoskeletal scaffolding. siRNA-mediated WASF3 knockdown was associated with significant decrease in invasion in 2D-Matrigel assays in U87 (16.4%, p&amp;lt;0.001), U251(27.3%, p&amp;lt;0.001) and patient-derived USC02 (25.7%, p&amp;lt;0.001) glioma cells. Additionally, 3D invasion assays showed reduced invasion of WASF3KD U87 gliomaspheres (41.1%, p=0.011) into the surrounding matrix. Finally, WASF3 overexpression in USC02s demonstrated rescue of invasive capacity lost with LINC03045 knockdown (p&amp;lt;0.01). Taken together, RNA-seq analysis of downstream effectors of LINC03045, coupled with tumor invasion studies establish downstream mediator WASF3 as a novel regulator of GBM invasive potential.