Abstract 1439: Asparagine stimulates growth and endoplasmic reticulum stress response in a fumarate hydratase-deficient renal cell carcinoma cell line

Abstract

Abstract Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a syndrome characterized by leiomyomas and an aggressive form of papillary kidney cancer. The genetic driver of HLRCC is a loss-of-function mutation of the Krebs cycle enzyme fumarate hydratase (FH), resulting in increased levels of the Krebs cycle intermediate fumarate, and resulting impaired oxidative phosphorylation. as well as in stabilization of HIF1α protein, which is the final common pathway of many different types of renal cell carcinomas (RCCs). To better understand the energy sources utilized by FH-deficient cells, we examined the nutrient requirements of FH-deficient RCC UOK262 (UOK262D) cell line in comparison to UOK262 cells transfected with an FH transgene (UOK262R). Experiments with selective addition of nonessential amino acids (NEAA) from the media revealed that both UOK262D and UOK262R cell lines required asparagine (A), but not other NEAA, for growth after 96 hours of incubation in the absence of glutamine (G). A (100 uM) and G (2 mM) demonstrated an approximately equal effect on the growth rate of both FH deficient and repleted cells, which was only observed after a minimum of 96 hrs of incubation. The growth effects of A and G were additive in both cell lines. While G increased the oxygen consumption rate (OCR) and decreased glycolysis (ECAR) in both cell lines, addition of A had no additive effect on both parameters, suggesting that G is used for energy production and A is required for biomass production. Similar effects of A and G, alone and in combination, on proliferation were seen in FH deficient and repleted UOK268 cell lines and in the SDH deficient and repleted UOK269 cells. Both A and G activated the mTOR pathway in both UOK262D and UOK262R cell lines as indicated by increased phosphorylation of mTOR, S6K and S6RP at 48h. However, mTOR pathway activation was not observed after A and/or G exposure in UOK268 or UOK269 pairs of cell lines. To determine the genomic response of UOK262 cell lines to A and/or G, we conducted RNASeq analysis in cells incubated for 96 hours to A, G, both or neither. In UOK262D cells, A+G, but not A or G individually, increased RNA levels of many genes associated with endoplasmic reticulum stress response. These gene expression changes were not seen in the FH repleted cell line UOK262R incubated in A+G. Therefore, although the growth effects of A+G in UOK262R and UOK262D cells were similar, and the effects on mTOR signaling pathway effects were similar between the two cell lines, the RNASeq experiment revealed a fundamentally different response to the combination of the 2 amino acids in the FH deficient cells vs. the FH repleted cells. In summary, we have identified asparagine as a key nutrient in HLRCC, establishing it as a potential target for a metabolically rational treatment approach. This research was supported by the Intramural Research Program of the National Cancer Institute, NIH. Citation Format: Rony Panarsky, Youfeng Yang, W. Marston Linehan, Jeffrey A. Moscow. Asparagine stimulates growth and endoplasmic reticulum stress response in a fumarate hydratase-deficient renal cell carcinoma cell line [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 1439.