59 GPNMB:CD44 Signaling Drives Tumor Progression in Translocation Renal Cell Carcinoma
Abstract Background MiT/TFE-rearranged, translocation renal cell carcinoma (tRCC) is characterized by gene fusions involving TFE3 or TFEB, and is a rare, aggressive non-ccRCC, often occurring in children and with few biomarkers or targeted therapy options. TFE3 is the most commonly translocated gene with ASPSCR1, PRCC and SFPQ as fusion partners. GPNMB (Glycoprotein non-metastatic melanoma protein B) is a canonical transcriptional target of MiT/TFE proteins expressed at low levels in normal tissues but upregulated in numerous MiT/TFE-driven tumors (e.g. tRCC), where it is associated with poor prognosis. GPNMB can also be cleaved at its ECD by metalloproteinases and secreted from the surface of cancer cells. Soluble GPNMB can thus be detected in the serum of cancer patients and function as a surrogate biomarker. CD44, the primary receptor for GPNMB, is a tumor-associated antigen linked to cancer invasion, metastases and stemness, and is associated with poor prognosis in many cancers. The aim of the current study was to characterize the biology of GPNMB and CD44 in pre-clinical models of tRCC Methods We used the following human cell line models: As in vitro pre-clinical companion models we analyzed cells with stable doxycycline-inducible expression of WT-TFE3, PRCC-TFE3, SFPQ-TFE3 and NONO-TFE3 fusions using a) the Flp-In T-RExTM system in HEK293 cells, b) rtTA3 (Tet-on) system in HK2 proximal tubular epithelial cells and c) patient derived UOK cells with TFE3-fusion transgenes, comparing them to UOK cells from ccRCC patients. We also generated a novel transgenic knock-in mouse expressing the SFPQ-TFE3 fusion. We crossed SFPQ-TFE3LSL knock-in mice to 1) KSP-Cadherin 16 Cre mice that express Cre recombinase in the distal tubular epithelial cells and collecting ducts of the kidney. 2) Tamoxifen-inducible, Pax8- ERTCre mice diffusely expressing Cre recombinase following kidney development. We also examined tumorigenesis in the previously described PRCC-TFE3LSL; Ksp-Cre mice (W. Marston Linehan, NIH), and 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). We are also currently examining blood serum, EDTA plasma and urine samples from non-metastatic and metastatic ccRCC and tRCC cases, obtained courtesy of the Kidney Cancer Program at Johns Hopkins. Results To further characterize GPNMB functionality and value as a cell-surface therapeutic target in tRCC, we engineered cell lines with genomic deletion of GPNMB via CRSIPR-Cas9 editing. Deletion of GPNMB in PRCC-TFE3 cell lines [UOK120/UOK124] significantly decreased clonogenic growth in 2D, spheroid size and viability and tumor xenograft growth in NSG mice and was associated with a decrease in phosphorylation of mTORC1 substrates [p-P70S6K, p-4EBP1]. We then examined expression of CD44, the primary receptor for GPNMB, and a tumor-associated antigen associated with poor prognosis in many cancers. Expression of CD44 and its ligand SPP1/OPN, was significantly increased in bulk RNA-Seq data from multiple transgenic models of SFPQ-TFE3, PRCC-TFE3 and ASPSCR1-TFE3, in human tRCC cases compared to normal kidney, and in SFPQ-TFE3/ PRCC-TFE3 transgenic kidney tumors and an ASPSCR-TFE3 PDX model, by immunoblotting and IHC, with increased membrane localization. shRNA-mediated depletion of CD44 profoundly and specifically decreased clonogenicity of multiple TFE3-fusion lines, with no effect seen in ccRCC lines. Conclusions In conclusion, GPNMB regulates the growth of tRCC cells, potentially via an autocrine mechanism involving its receptor CD44, and targeting GPNMB-CD44 signaling may be of therapeutic benefit in tRCC. DOD CDMRP Funding yes