Abstract 3109: Investigating the role of wild-type TFE3 in renal cell carcinoma cells harboring TFE3 fusions with spliceosome machinery associated genes

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Abstract Background: Translocation Renal Cell Carcinoma (tRCC) represents an aggressive subtype of kidney cancer associated with various gene fusions involving translocation of one of two members of micropthalmia transcription factor (MiT) family, TFE3 or TFEB. Despite the identification of multiple TFE3 gene fusions in tRCC, heterogeneous phenotype and various dysregulated signaling pathways resulting from variety of gene fusion partners pose a challenge to establish effective treatments for these patients. In this work, we assessed the role of wild type TFE3 (wt-TFE3) in RCC cell bearing TFE3 fusion with genes associated with pre-mRNA splicing factor machinery (NONO, SFPQ and PRCC).Methods: Endogenous SFPQ-TFE3 expressing cells, RP-RO7, were generated from patient derived xenograft established in NSG mice. UOK-109 and UOK-146 (kindly provided by Dr. Marston Lenehan, NCI) were used as endogenous NONO-TFE3 and PRCC-TFE3 expressing cells, respectively. RNA interference (RNAi) mediated knockdown with differential exon targeting strategy was employed to study wt-TFE3 loss of function in RP-R07, UOK-109 and UOK-146, respectively. Real time monitoring of cell viability assay with multiplex readout were developed to investigate the effect of wt-TFE3 loss of function in 2D and 3D culture system. Two different 3D models were used;1) Tumor growth model, in which cancer cell interaction with extracellular matrix and stromal cells were represented in a multiculture-spheroid system; 2) Invasion model, in which cell ability to invade basement membrane barrier was modeled with matrix restricted spheroid, and the invasion trajectories were observed and quantified. Exogenous expression of wt-TFE3-EGFP was utilized to study the protein subcellular localization in RP-R07, UOK-109 and UOK-146 2D cultures and 3D culture system.Results: Consistent with the expression of chimeric TFE3, wt-TFE3-EGFP ectopic expression demonstrated strong nuclear localization in RP-R07, UOK-109 and UOK-146. Multiplex readout of cells viability assay showed that transient loss of wt-TFE3 in RP-R07, UOK-109 and UOK-146 curtails the proliferation rate of these cells in 2D and 3D models in fusion partner dependent manner (P<0.05). 3D invasion assay coupled with RNAi strategy in RP-R07, UOK-109 and UOK-146 demonstrated the role wt-TFE3 in invasion potential of these cell in fusion partner dependent fashion (P<0.05). Downstream analysis suggested that the wt-TFE3 transient silencing affected cell proliferation and invasion ability via inhibition of the IRS-PI3K-mTOR axis.Conclusion: Our results suggest the potential role of wt-TFE3 in tRCC oncogenesis irrespective of its different fusion partner. Citation Format: Nur P. Damayanti, Khunsha Ahmed, May Elbanna, Chinghai Kao, Roberto Pili. Investigating the role of wild-type TFE3 in renal cell carcinoma cells harboring TFE3 fusions with spliceosome machinery associated genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3109.