73 An EGFR-family Signaling Axis Confers Therapeutic Vulnerability in Translocation RCC

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Abstract Background Translocation renal cell carcinoma (tRCC) and perivascular epithelioid cell tumors (PEComas) are a rare subtype of non-clear cell, sporadic kidney cancer driven by chromosomal translocations involving the MiT/TFE family of transcription factors (TFE3 [Xp11.23], TFEB [6p21.1], and MITF [3p13]), which are key regulators of lysosomal biogenesis. The resulting fusions of MiT/TFE genes with various partner genes (the most common being ASPSCR1, PRCC, and SFPQ), leads to constitutive nuclear localization and activation of the chimeric transcription factors. tRCC is a common renal carcinoma subtype occurring in children, representing up to a third of all cases, and though rarer in adults, it is frequently aggressive and more common in females. Notably, tRCC exhibits a high degree of morphologic and clinical heterogeneity, at least in part due to varying TFE3 fusion partners, and their molecular landscape has only been partially defined, prompting an urgent need to identify biomarkers and therapeutic targets. Methods A) Human cell line models: As in vitro pre-clinical companion models we analyzed patient derived UOK cells with TFE3-fusion transgenes, comparing them to UOK cells from ccRCC patients. B) Murine transgenic models: (1) We generated a novel transgenic knock-in mouse expressing the human SFPQ-TFE3 fusion. We crossed SFPQ-TFE3LSL knock-in mice to KSP-Cadherin 16 Cre mice that express Cre recombinase in the distal tubular epithelial cells and collecting ducts of the kidney, and Tamoxifen-inducible, Pax8-ERTCre mice diffusely expressing Cre recombinase following kidney development. (2) We also examined tumorigenesis in the previously described PRCC-TFE3LSL; Ksp-Cre mice (W. Marston Linehan, NIH), and (3) in an ASPSCR1-TFE3 PDX mouse model and a LN metastatic lesion from a patient with the ASPSCR1-TFE3 fusion (Dr. John Copland, Mayo Clinic). Results EGFR activation in tRCC: Germline variations in MITF and associated papillary RCC have been linked with somatic gains in chromosome 7 and EGFR amplification, and in a previously published transgenic model of PRCC-TFE3LSL; Ksp-Cre (PTK)-driven renal tumorigenesis, GSEA analyses showed enrichment of genes associated with EGFR activation. Therefore, we examined EGFR signaling at the protein level in this model and found that total and membrane EGFR expression and downstream ERK activation were increased in tumor lysates, FFPE specimens and primary renal cells by immunoblotting and IHC compared to controls. Levels of phosphorylated and total EGFR, HER2 and HER3 were also elevated in tamoxifen-treated, SFPQ-TFE3 Pax8-ERTCre mice compared to controls. UOK cells with TFE3 fusions [UOK120,124,146=PRCC-TFE3; UOK109=NONO-TFE3 and UOK145=SFPQ-TFE3] showed upregulated expression of ERBB3/ERBB4 mRNA expression by RNAseq, compared to ccRCC controls [A498, UOK111, UOK140, UOK150]. Correspondingly, tRCC cell lines also demonstrated increased p-HER3 using a Proteome Profiler Human Phospho-RTK Array Kit and increased p-EGFR, p-HER2 and p-HER3 by immunoblotting and immunofluorescence of starved lysates, compared to ccRCC controls. EGFR and HER2 surface protein expression was also increased in tumor xenografts of PRCC-TFE3 cells [UOK146 and UOK124] established in NSG (NOD-scid gamma) mice, and in tumors derived from an ASPSCR1-TFE3 patient grown in NSG mice [ samples courtesy, of Dr John Copland, Mayo Clinic, FL]. Treatment with EGFR/HER2/HER3 kinase inhibitors Afatinib, Lapatinib and Erlotinib selectively decreased the clonogenicity, induced potent cytotoxicity and decreased ERK-mTOR signaling in tRCC cell lines, with no effect observed on ccRCC cells. Similar effects were observed in tRCC cell lines transiently treated with EGFR, HER2 or HER3 shRNA. Conclusions Studies are currently ongoing to determine the efficacy of EGFR inhibitors in reversing kidney tumor burden or xenograft growth in the murine transgenic and human cell line models, respectively. Thus, EGFR family expression and/or activity may represent a novel category of biomarkers, and EGFR inhibitors may potentially be leveraged for the treatment of tRCC. DOD CDMRP Funding yes

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