Abstract 3705: L-2HG, oncometabolite-driven epigenetic and epitranscriptomic reprogramming creates metabolic vulnerability in renal cancer
Authors: Kundu Anirban , Brinkley Garrett J. , Nam Hyeyoung , Karki Suman , Kirkman Richard , Widden Hayley , Johnson Michelle , Liu Juan , Heidarian Yasaman , Mahmoudzadeh Nader , Absher Devin , Ding Han-Fei , Crosman David , Placzek William J. , Locasale Jason , Rakheja Dinesh , Darley-Usmar Victor , Tennessen Jason , Sudarshan Sunil 
Published: April 2023

The oncometabolite, L-2-hydroxyglutarate (L-2HG) is elevated in the most common form of renal cell carcinoma-RCC (clear cell histology) and promotes tumor progression. L-2HG is structurally similar to α-ketoglutarate (α-KG). Therefore, L-2HG can competitively inhibit enzymes that utilize α-KG as a cofactor including α-KG-dependent dioxygenases that can profoundly impact gene expression via effects on the epigenome and epitranscriptome. RCC cell lines lack the L-2HG dehydrogenase enzyme (L2HGDH), resulting in their high L-2HG level. RNA-seq of control (high L-2GH) and an L2HGDH reconstituted (low L-2HG) RCC cell line has revealed that L-2HG suppresses the expression of serine biosynthesis genes, PHGDH and PSAT1. The findings were consistent in the patient samples where high L-2HG renal tumors had lower levels of PHGDH and PSAT1 expressions than that of the low L-2HG renal tumors and the patient-matched normal kidneys. Consistently, 13C-metabolomics labeling studies demonstrate that raised L-2HG suppresses de novo serine biosynthesis. Moreover, LC-MS analysis of the metabolites isolated from the kidneys of L2HGDH KO and wild-type (WT) mice revealed less serine content in the absence of L2HGDH, further confirming that high L-2HG suppresses serine biosynthesis in vivo. We found that L-2HG-mediated inhibition of the α-KG-dependent histone demethylase KDM4C silences ATF4 transcription. ATF4 is a master regulator of amino acid biosynthetic genes including PHGDH and PSAT1. Using ATF4 gain of function analysis, we confirmed that high L-2HG causes the suppression of PHGDH and PSAT1 in an ATF4-dependent manner. In addition, we demonstrate that L-2HG promotes the accumulation of the epitranscriptomic mark N⁶-methyladenosine (m6A) via inhibiting α-KG-dependent RNA demethylases ALKBH5 and FTO. In the setting of high L-2HG, m6A is enriched in the 3’-UTR region of transcripts including PSAT1. Using mutational analysis, we demonstrate that L-2HG promotes m6A accumulation at a specific site within the 3’UTR of PSAT1 that silences its translation. In accord with these data, found that high L-2HG RCC cells require exogenous serine for in vitro proliferation and in vivo tumor growth. Furthermore, this serine liability can be rescued upon lowering cellular L-2HG levels. Metabolomics analyses demonstrate that exogenous serine is required to maintain cellular pools of glutathione in high L-2HG RCC which supports both proliferation and resistance to oxidative stress. The data indicate that the L-2HG elevation in RCC reconfigures tumor metabolism through a bimodal mechanism via remodeling of both the epigenome and epitranscriptome. This results in a serine liability in the setting of raised L-2HG. Collectively, our data unmask a metabolic vulnerability that can be harnessed for precision-based approaches to kidney cancer.

Citation Format: Anirban Kundu, Garrett J. Brinkley, Hyeyoung Nam, Suman Karki, Richard Kirkman, Hayley Widden, Michelle Johnson, Juan Liu, Yasaman Heidarian, Nader Mahmoudzadeh, Devin Absher, Han-Fei Ding, David Crosman, William J. Placzek, Jason Locasale, Dinesh Rakheja, Victor Darley-Usmar, Jason Tennessen, Sunil Sudarshan. L-2HG, oncometabolite-driven epigenetic and epitranscriptomic reprogramming creates metabolic vulnerability in renal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3705.