Introduction: The oncometabolite L-2-hydroxyglutarate (L-2HG) is elevated in a large percentage of the most common form of RCC (clear cell histology) and promotes tumor progression. Structurally similar to a-ketoglutarate (a-KG), L-2HG can competitively inhibit enzymes that utilize a-KG as a cofactor including a-KG-dependent dioxygenases that can profoundly impact gene expression via effects on the epigenome and epitranscriptome. Methods: Patient samples, cell lines, Crisper-Cas9 L2HGDH knock out whole body C57-B6 mice, and xenograft models of nude mice were used. Data were analyzed via real-time PCR, western blot, luciferase assays, untargeted metabolomics, and [U-13C] Glucose labeling Results: RCC cell lines lack the L-2HG dehydrogenase enzyme (L2HGDH) which results in their high L-2HG levels. RNA-seq of control (high L-2GH) and an L2HGDH reconstituted (low L-2HG) RCC cell line revealed that L-2HG suppresses the expression of serine biosynthesis genes, PHGDH and PSAT1. Furthermore, patient samples with high L-2HG renal tumors had lower levels of PHGDH and PSAT1 expressions (both mRNA and protein) than that of the low L-2HG renal tumors and the patient-matched normal kidneys. In vitro 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 mice revealed less serine content in the absence of L2HGDH. In addition, we demonstrate that L-2HG promotes the accumulation of the epitranscriptomic mark N6-methyladenosine (m6A) via inhibiting a-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. In accord with these data, high L-2HG RCC cells require exogenous serine for in vitro proliferation and in vivo tumor growth. 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. Conclusions: 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. SOURCE OF Funding: The research reported in this article was supported by the National Institutes of Health (R01CA200653 and I01BX002930 to S.S.; F30CA232397 and T32GM008361 to G.B.) and in part by the University of Alabama at Birmingham O’Neal Comprehensive Cancer Center (P30CA013148). Tumor genotyping was supported in part by the University of Texas Health Science Center at San Antonio Mays Cancer Center Genomics Shared Resource Facility (P30CA054174). J.M.T. is supported by a Maximizing Investigators’ Research Award from the National Institute of General Medical Sciences (R35GM119557).
