Through unbiased metabolomics, we identified elevations of the metabolite 2-hydroxyglutarate (2HG) in renal cell carcinoma (RCC). 2HG can inhibit 2-oxoglutaratre (2-OG) dependent dioxygenases which mediate epigenetic events including DNA and histone demethylation. 2HG accumulation, specifically the D- enantiomer, can result from gain of function mutations of isocitrate dehydrogenase (IDH1, IDH2) found in several different tumors. In contrast, kidney tumors demonstrate elevations of the L enantiomer of 2HG (L-2HG). High 2HG tumors demonstrate reduced DNA levels of 5-hydroxymethylcytosine (5hmC) consistent with 2-HG mediated inhibition of TET (Ten Eleven Translocation) enzymes which convert 5-methylcystoine (5mC) to 5hmC. L-2HG elevation is mediated in part by reduced expression of L-2HG dehydrogenase (L2HGDH). L2HGDH reconstitution in RCC cells lowers L-2HG and promotes 5hmC accumulation. Additionally, L2HGDH expression in RCC cells reduces histone methylation and suppresses in vitro tumor phenotypes. Our report identifies L-2HG as an epigenetic modifier and putative oncometabolite in kidney cancer.

Elevation of L-2-hydroxylgutarate (L-2-HG) in renal cell carcinoma (RCC) is due in part to reduced expression of L-2-HG dehydrogenase (L2HGDH). However, the contribution of L-2-HG to renal carcinogenesis and insight into the biochemistry and targets of this small molecule remains to be elucidated. Genetic and pharmacologic approaches to modulate L-2-HG levels were assessed for effects on and phenotypes. Metabolomics was used to dissect the biochemical mechanisms that promote L-2-HG accumulation in RCC cells. Transcriptomic analysis was utilized to identify relevant targets of L-2-HG. Finally, bioinformatic and metabolomic analyses were used to assess the L-2-HG/L2HGDH axis as a function of patient outcome and cancer progression. L2HGDH suppresses both cell migration and tumor growth and these effects are mediated by L2HGDH's catalytic activity. Biochemical studies indicate that glutamine is the predominant carbon source for L-2-HG via the activity of malate dehydrogenase 2 (MDH2). Inhibition of the glutamine-MDH2 axis suppresses phenotypes in an L-2-HG-dependent manner. Moreover, growth of RCC cells with basal elevation of L-2-HG is suppressed by glutaminase inhibition. Transcriptomic and functional analyses demonstrate that the histone demethylase KDM6A is a target of L-2-HG in RCC. Finally, increased L-2-HG levels, copy loss, and lower L2HGDH expression are associated with tumor progression and/or worsened prognosis in patients with RCC. Collectively, our studies provide biochemical and mechanistic insight into the biology of this small molecule and provide new opportunities for treating L-2-HG-driven kidney cancers.

Background: The D-enantiomer of 2-hydroxygultarate (D-2HG), along with fumarate and succinate, are considered oncometabolites that promote tumorigenesis. Our lab has reported elevated levels of the L-enantiomer of 2-hydroxyglutare (L-2HG) in clear cell renal cell carcinoma (ccRCC), in part due to reduced expression of the enzyme L-2HG dehydrogenase (L2HGDH). Objectives: Here we evaluated the underlying biochemical mechanisms of L-2HG accumulation and characterized the contribution of the L-2HG/L2HGDH axis to tumorigenesis. Experimental strategy: We assessed the role of raised L-2HG in renal carcinogenesis via both in vitro and in vivo means. Mutagenesis and knock down approaches were applied to study involvement of loss of L2HGDH in accumulation of high L-2HG levels and associated tumor phenotypes. Metabolomics coupled with 13C tracer labeling studies were utilized to dissect the biochemical axis that promotes L-2HG accumulation in RCC cells. Results: Translational based studies demonstrate that loss of L2HGDH expression is associated with both cancer progression and worsened outcomes. In connection with our previous findings, we demonstrate that raising cellular L-2HG levels by treatment with cell permeable octyl ester of L-2HG as well as by shRNA-mediated knock down of L2HGDH in renal epithelial cells (HK-2) promotes in vitro tumor phenotypes. Concurrently, the epigenetic mark 5hmc was significantly decreased under high L-2HG levels. Further investigation with restoration of L2HGDH in RCC cells (RXF393 and A498) shows decreased L-2HG levels and suppression of in vivo tumor growth in nude mice (NU/NU). Interestingly, expression of loss-of-function mutant of L2HGDH was unable to decrease L-2HG levels and failed to suppress in vitro and in vivo tumor phenotypes. In addition, high L-2HG levels were found to upregulate epithelial-mesenchymal-transition (EMT) marker SNAIL1 and correspondingly downregulate E-cadherin. Biochemical studies demonstrate that the predominant carbon source for L-2HG in RCC is glutamine through the activity of glutaminase and malate dehydrogenase (MDH). Pharmacological inhibition of the glutamine/MDH axis by treatment of glutaminase inhibitor (CB-839) and MDH inhibitor (4k) in RCC cells reduced L-2HG levels and mitigates in vitro tumor phenotypes. Furthermore, suppression of in vitro phenotypes by CB-839 and shRNA-mediated MDH2 knockdown was rescued by concurrent treatment with octyl ester of L-2HG. Finally, restoration of L2HGDH promoted the expression of genes targeted by the polycomb repressor complex 2 (PRC2), whereas inhibition of PRC2 in high L-2HG cells suppressed tumor phenotypes. Conclusion: Collectively, our data demonstrate the biologic relevance of high L-2HG to renal carcinogenesis and reveal novel therapeutic opportunities for L-2HG driven kidney tumors. Citation Format: Sandeep Balu Shelar, Eun-hee Shim, Garrett Brinkley, Anirban Kundu, Hyeyoung Nam, Francesca Carobbio, Tyler Poston, Jubilee Tan, Daniel Benson, Dinesh Rakheja, Richard Kirkman, Yusuke Sato, Seishi Ogawa, Shilpa Dutta, Sadanandan E. Velu, David Crossman, Anja Becker, Conrad Kunick, Sunil Sudarshan. L-2HG/ L2HGDH axis as therapeutic target for kidney cancer [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 5482.

<p>Supplementary Figure 1. A498 cells were stable transduced with the indicated construct. Protein lysates were harvested and immunoblotted for the indicated proteins.</p>

<p>Supplementary Figure 3. HEK293 cells were transiently transfected with either CV or L2HGDH cDNA and TET1 cDNA. Protein lysates were harvested and immunoblotted for the indicated proteins.</p>