Combination of Tipifarnib and Sunitinib Overcomes Renal Cell Carcinoma Resistance to Tyrosine Kinase Inhibitors via Tumor-Derived Exosome and T Cell Modulation

Simple Summary Metastatic renal cell carcinoma continues to have a poor prognosis. Chemotherapies and immuno-oncologic therapies have garnered increasing importance in cancer therapy, with improvements in patient care and survival. However, a large proportion of patients present with tumors resistant to these treatments. Exosomes are small extracellular vesicles secreted by all nucleated cells that have proven to be key actors in this resistance. Exosomes carry bioactive oncogenic cargos that reprogram target cells to promote tumor growth, migration, metastasis, immune evasion, and chemotherapy resistance. Tipifarnib, in combination with standard therapy, decreased tumor growth in the setting of chemotherapeutic resistance through an exosome-mediated mechanism. After using a qNANO IZON system to compare tumor-derived exosomes collected from untreated and tipifarnib-treated cells, all cancerous cell lines displayed a reduction of vesicle concentration. Tipifarnib also directly inhibited PD-L1 protein expression in chemo-sensitive cell lines and resistant cell lines. Background: Tyrosine kinase inhibitors (TKI) were initially demonstrated as an efficacious treatment for renal cell carcinoma (RCC). However, after a median treatment length of 14 months, a vast majority of patients develop resistance. This study analyzed a combination therapy of tipifarnib (Tipi) + sunitinib that targeted exosome-conferred drug resistance. Methods: 786-O, 786-O-SR (sunitinib resistant), A498, A498-SR, Caki-2, Caki-2-SR, and 293T cells were cultured. Exosomes were collected using differential ultracentrifugation. Cell proliferation, Jurkat T cell immune assay, and immunoblot analysis were used for downstream analysis. Results: SR exosomes treatment displayed a cytotoxic effect on immune cells. This cytotoxic effect was associated with increased expression of PD-L1 on SR exosomes when compared to sunitinib-sensitive (SS) exosomes. Additionally, Tipi treatment downregulated PD-L1 expression on exosomes derived from SR cell lines. Tipi's ability to downregulate PD-L1 in exosomes has a significant application within patients. Exosomes collected from patients with RCC showed increased PD-L1 expression over subjects without RCC. Next, exosome concentrations were then compared after Tipi treatment, with all SS cell lines displaying an even greater reduction. On immunoblot assay, 293T cells showed a dose-dependent increase in Alix with no change in either nSMase or Rab27a. Conversely, all the SS and SR cell lines displayed a decrease in all three markers. After a cell proliferation employed a 48-h treatment on all SS and SR cell lines, the drug combination displayed synergistic ability to decrease tumor growth. Conclusions: Tipifarnib attenuates both the exosome endosomal sorting complex required for endosomal sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways, thereby blocking exosome biogenesis and secretion as well as downregulating PD-L1 on SS and SR cells.