Abstract 16722: L-2-Hydroxyglutarate Protects Against Myocardial Ischemia-Reperfusion Injury

Background We have found L-2-hydroxyglutarate dehydrogenase (L2HGDH) to be downregulated in the myocardium of mice subjected to transverse aortic constriction (TAC). L2HGDH is an important regulator of mitochondrial bioenergetics by catalyzing the conversion of L-2-hydroxyglutarate (L2-HG) to α-ketoglutarate. However, the connection between L2-HG accumulation and heart failure is not yet understood. Purpose Purpose of our study was to investigate the role of increased L2-HG levels in heart failure and the potential role of L2HGDH overexpression as therapeutic strategy. Methods For in vitro studies, primary rat neonatal cardiomyocytes (NRVCMs) were incubated with L2-HG. L2HGDH was overexpressed using adeno-associated virus (AAV) 6 vectors. Mitochondrial membrane potential was measured using TMRE (tetramethylrhodamine ethyl ester) dye. Mitochondrial reactive oxygen species production was monitored using MitoSOX. We further determined activation of fetal gene program by real time qPCR and macrophage migration using RAW 264.7 cells and transwell inserts. mTOR activation was analyzed by Western blot with antibodies against phosphorylated mTOR and ribosomal protein S6. AAV9 expressing L2HGDH or luciferase was injected in C57BL/6N mice two weeks prior to TAC and heart function was monitored by echocardiography for 6 weeks. Results L2-HG acts as a pro-hypertrophic stimulus in NRVCMs as shown by upregulation of a fetal gene expression pattern and an increase in cardiomyocyte cross-sectional area upon L2-HG treatment. Furthermore, mRNA levels of macrophage chemoattractant protein 1 were increased in L2-HG treated cells, which correlated with enhanced macrophage migration towards supernatant of L2-HG treated NRVCMs. Furthermore, we could confirm that L2-HG augmented mTOR signaling by affecting the phosphorylation status of ribosomal protein S6. AAV-mediated L2HGDH overexpression in NRVCMs led to a significant 2.1-fold decrease in the accumulation of ROS production. Moreover, we found an inhibition of endothelin-1 induced mitochondrial membrane depolarization in AAV6-L2HGDH transduced cells. Pretreatment of mice with AAV9-L2HGDH prior to TAC resulted in significantly reduced heart weight to tibia length ratios (HW/TL) and cardiomyocyte area. Importantly, heart function was notably improved in mice receiving gene therapy (ejection fraction, EF: 36.18±6.63%, fractional shortening, FS: 16.72±4.01%) whereas control animals showed marked decline in myocardial contractility (EF: 20.14±8.24%, FS: 12.66±6.66%). Conclusion L2-HG causes cardiomyocyte dysfunction by activating mTOR signaling pathway, a well-characterized critical inducer of myocyte hypertrophy, and enhancing macrophage migration, leading to establishment of a pro-inflammatory environment in the myocardium. Moreover, our results point out towards a novel preventive approach for cardiac hypertrophy and heart failure by cardiomyocyte-specific L2HGDH overexpression. Acknowledgement/Funding DZHK (Deutsches Zentrum für Herz-Kreislaufforschung)

Cardiac hypertrophy is a common response to hemodynamic stress in the heart and can progress to heart failure. To investigate whether the transcription factor cardiovascular basic helix-loop-helix factor 1/hairy/enhancer of split related with YRPW motif 2 (CHF1/Hey2) influences the development of cardiac hypertrophy and progression to heart failure under conditions of pressure overload, we performed aortic constriction on 12-wk-old male wild-type (WT) and heterozygous (HET) mice globally underexpressing CHF1/Hey2. After aortic banding, WT and HET mice showed increased cardiac hypertrophy as measured by gravimetric analysis, as expected. CHF1/Hey2 HET mice, however, demonstrated a greater increase in the ventricular weight-to-body weight ratio compared with WT mice (P < 0.05). Echocardiographic measurements showed a significantly decreased ejection fraction compared with WT mice (P < 0.05). Histological examination of Masson trichrome-stained heart tissue demonstrated extensive fibrosis in HET mice compared with WT mice. TUNEL staining demonstrated increased apoptosis in HET hearts (P < 0.05). Exposure of cultured neonatal myocytes from WT and HET mice to H(2)O(2) and tunicamycin, known inducers of apoptosis that work through different mechanisms, demonstrated significantly increased apoptosis in HET cells compared with WT cells (P < 0.05). Expression of Bid, a downstream activator of the mitochondrial death pathway, was expressed in HET hearts at increased levels after aortic banding. Expression of GATA4, a transcriptional activator of cardiac hypertrophy, was also increased in HET hearts, as was phosphorylation of GATA4 at Ser(105). Our findings demonstrate that CHF1/Hey2 expression levels influence hypertrophy and the progression to heart failure in response to pressure overload through modulation of apoptosis and GATA4 activity.

Previously, the Sudarshan laboratory has identified elevations in the level of the oncometabolite L-2-hydroxyglutarate (L-2HG) in human renal cell carcinomas (RCCs). Of particular interest in these regards, both L-2HG inhibits 2-oxoglutarate-dependent dioxygenases (2-OGDs), including histone demethylases, which regulate the epigenetic landscape of cells, and cellular differentiation. Thus, it was of interest to determine whether the accumulation of L-2HG alters the differentiation of the normal renal proximal tubule (RPT), ultimately affecting the phenotype of the RCC. Towards these ends, L-2HG dehydrogenase (L2HGDH) was knocked down in primary cultures of normal rabbit RPT cells, and their capacity for cellular differentiation was examined. Initially, we examined the ability of primary RPT cells to form tubules in matrigel following an L2HGDH knockdown (KD). While tubulogenesis was stimulated by Epidermal Growth Factor (EGF) in primary RPT cells transduced with a control lentiviral vector, tubulogenesis in matrigel was dramatically impaired in primary RPT cells when L2HGDH was knocked down by lentiviral L2HGDH shRNA. In order to determine whether the expression of differentiated transport functions was affected, RealTime PCR (RTPCR) was conducted. The results indicated that an L2HGDH knockdown (80%) resulted in a reduction in the expression of the Na+/Pi cotransporter NaPi2a (81%), the Na+/glucose cotransporter SGLT2 (88%), the water transporter Aquaporin 1 (AQP1) (95%), and the Na,K-ATPase ß1 subunit (atp1b1) (43%), whereas the expression of the p-Aminohippurate transporter OAT1 was not significantly affected. Similar results were obtained when using L2HGDH siRNA and lentiviral shRNA. Not only is tissue architecture critical for the maintenance of functional differentiation, but alterations in tissue architecture are a necessary component of tumor formation. Thus, we are examining the underlying causes for the reduced tubulogenesis in matrigel cultures with an L2HGDH KD. In our initial studies, we examined the expression of differentiated transport functions in matrigel vs. monolayer cultures. Notably, while the expression such transporters as AQP2 increased dramatically in matrigel (as opposed to monolayer cultures), the expression of other transporters such as NaPi2a, SGLT2 and atp1b1 was affected to a much smaller extent. Of particular interest in these regards, is the known role of AQP1 in the migration of renal proximal tubule cells, tumor spread, and wound healing. We are currently studying the effects of an L2HGDH KD on the expression of such genes as AQP1 during tubulogenesis in matrigel.This work has been funded by Grant # 1RO1CA200653-01A1 to Dr. Sunil Sudarshan (PI) with a subaward to Dr. Mary Taub. Citation Format: Mary L. Taub, Sunil Sudarshan. Oncometabolite L-2-hydroxyglutarate blocks differentiation of renal proximal tubule cells in matrigel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4364.

l-2-Hydroxyglutarate aciduria (L-2-HGA) is an autosomal recessive neurometabolic disorder caused by a mutation in the l-2-hydroxyglutarate dehydrogenase (L2HGDH) gene. In this study, we generated L2hgdh knockout (KO) mice and observed a robust increase of l-2-hydroxyglutarate (L-2-HG) levels in multiple tissues. The highest levels of L-2-HG were observed in the brain and testis, with a corresponding increase in histone methylation in these tissues. L2hgdh KO mice exhibit white matter abnormalities, extensive gliosis, microglia-mediated neuroinflammation, and an expansion of oligodendrocyte progenitor cells (OPCs). Moreover, L2hgdh deficiency leads to impaired adult hippocampal neurogenesis and late-onset neurodegeneration in mouse brains. Our data provide in vivo evidence that L2hgdh mutation leads to L-2-HG accumulation, leukoencephalopathy, and neurodegeneration in mice, thereby offering new insights into the pathophysiology of L-2-HGA in humans.

L-2-hydroxyglutarate (L2HG) couples mitochondrial and cytoplasmic energy metabolism to support cellular redox homeostasis. Under oxygen-limiting conditions, mammalian cells generate L2HG to counteract the adverse effects of reductive stress induced by hypoxia. Very little is known, however, about whether and how L2HG provides tissue protection from redox stress during low-flow ischemia (LFI) and ischemia-reperfusion injury. We examined the cardioprotective effects of L2HG accumulation against LFI and ischemia-reperfusion injury and its underlying mechanism using genetic mouse models. L2HG accumulation was induced by homozygous (L2HGDH [L-2-hydroxyglutarate dehydrogenase]-/-) or heterozygous (L2HGDH+/-) deletion of the L2HGDH gene in mice. Hearts isolated from these mice and their wild-type littermates (L2HGDH+/+) were subjected to baseline perfusion and 90-minute LFI or 30-minute no-flow ischemia followed by 60- or 120-minute reperfusion. Using [13C]- and [31P]-NMR (nuclear magnetic resonance) spectroscopy, high-performance liquid chromatography, reverse transcription quantitative reverse transcription polymerase chain reaction, ELISA, triphenyltetrazolium staining, colorimetric/fluorometric spectroscopy, and echocardiography, we found that L2HGDH deletion induces L2HG accumulation at baseline and under stress conditions with significant functional consequences. In response to LFI or ischemia-reperfusion, L2HG accumulation shifts glucose flux from glycolysis towards the pentose phosphate pathway. These key metabolic changes were accompanied by enhanced cellular reducing potential, increased elimination of reactive oxygen species, attenuated oxidative injury and myocardial infarction, preserved cellular energy state, and improved cardiac function in both L2HGDH-/- and L2HGDH+/- hearts compared with L2HGDH+/+ hearts under ischemic stress conditions. L2HGDH deletion-induced L2HG accumulation protects against myocardial injury during LFI and ischemia-reperfusion through a metabolic shift of glucose flux from glycolysis towards the pentose phosphate pathway. L2HG offers a novel mechanism for eliminating reactive oxygen species from myocardial tissue, mitigating redox stress, reducing myocardial infarct size, and preserving high-energy phosphates and cardiac function. Targeting L2HG levels through L2HGDH activity may serve as a new therapeutic strategy for cardiovascular diseases related to oxidative injury.

Renal L-2-hydroxyglutarate dehydrogenase activity promotes hypoxia tolerance and mitochondrial metabolism in Drosophila melanogaster

Myelodysplastic Syndromes: A More Global 5-Hydroxymethylcytosine Deficiency Disorder Than Suggested By the Presence of TET2 Mutations

The L-2-hydroxyglutarate dehydrogenase (L2HGDH) gene encodes an important mitochondrial enzyme. However, its altered activity results in excessive levels of L-2-hydroxyglutarate, which results in diverse psychiatric features of intellectual disability. In the current study, we executed an in-silico analysis of all reported L2HGDH missense and nonsense variants in order to investigate their biological significance. Among the superimposed 3D models, the highest similarity index for a wild-type structure was shown by the mutant Glu336Lys (87.26%), while the lowest similarity index value was shown by Arg70* (10.00%). Three large active site pockets were determined using protein active site prediction, in which the 2nd largest pocket was shown to encompass the substrate L-2-hydroxyglutarate (L2HG) binding residues, i.e., 89Gln, 195Tyr, 402Ala, 403Gly and 404Val. Moreover, interactions of wild-type and mutant L2HGDH variants with the close functional interactor D2HGDH protein resulted in alterations in the position, number and nature of networking residues. We observed that the binding of L2HG with the L2HGDH enzyme is affected by the nature of the amino acid substitution, as well as the number and nature of bonds between the substrate and protein molecule, which are able to affect its biological activity.

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.

Renal cell carcinoma (RCC) is among the top 10 cancers in the USA. Despite several approved therapies, patients with the advanced disease rarely have durable responses and therefore, face a poor prognosis (median survival 2-3 years). This underscores the need for new strategies. Alterations in metabolism are well-established in cancers including RCC. The oncometabolite, L-2-hydroxyglutarate (L-2HG) is elevated in the most common form of RCC (clear cell histology) and promotes tumor progression. However, L-2HG’s roles in RCC progression and its mediated therapeutic vulnerability are yet to be explored. RCC cell lines lack the L-2HG dehydrogenase enzyme (L2HGDH) which results in their high L-2HG level. RNA-seq of control (high L-2GH) and an L2HGDH reconstituted (low L-2HG) RCC cell line reveals that L-2HG suppresses the expression of serine biosynthesis genes, PHGDH and PSAT1. In agreement, high L-2HG renal tumors demonstrate lower levels of serine biosynthesis enzymes compared to their matched normal kidneys. Mechanistic studies reveal L-2HG-mediated remodeling of both the epigenome and epitranscriptome suppress serine biosynthesis genes. 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 lower serine levels in L2hgdh KO kidneys. In accordance with these data, found that high L-2HG RCC cells require exogenous serine for in vitro proliferation and in vivo tumor growth. Likewise, the pharmacologic blockade of serine uptake decreases the proliferation of high L-2HG RCC cells. Furthermore, this serine liability can be rescued upon lowering cellular L-2HG levels. Untargeted metabolomics analyses demonstrate that exogenous serine is required to maintain cellular pools of glutathione (GSH+GSSG) in high L-2HG RCC. This is particularly relevant as glutathione is among the most highly enriched metabolites in RCC compared to normal kidneys. Our metabolomics data also suggest that serine might be essential for the transsulfuration process of glutathione biosynthesis in RCC that lacks the xCT system required to uptake cysteine for transsulfuration. In vivo, we find that intratumoral levels of glutathione are reduced in mice fed a chow diet lacking serine compared to regular chow diet-fed mice. Pharmacologic inhibition of glutathione synthesis ablates the growth of high L-2HG RCC cells even in the presence of serine, suggesting the importance of redox homeostasis for RCC proliferation. The data indicate that the L-2HG elevation in RCC reconfigures tumor metabolism, resulting in serine liability. 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, Devin Absher, William J. Placzek, Jason Locasale, Dinesh Rakheja, Victor Darley-Usmarc, Jason Tennessen, Sunil Sudarshan. Metabolic liabilities in high L-2HG kidney cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B005.

H3K27M-mutant diffuse midline glioma (DMG) patients have no proven effective therapies beyond radiation. ONC201, a mitochondrial protease ClpP agonist, has recently demonstrated efficacy in these patients, but the mechanism behind this remains unknown. We assessed clinical outcomes, tumor sequencing, and tissue samples from patients treated in two completed multi-site clinical studies (n=71). Patients treated with ONC201 monotherapy following initial radiation but prior to recurrence (n=35) demonstrated a median overall survival of 21.7 months and a median progression-free survival of 12.2 months. Radiographic response was associated with increased expression of key tricarboxylic acid cycle-related genes in baseline tumor sequencing. Similar survival benefits were observed using in utero electroporation and orthotopic mouse models of H3K27M-DMG (median 107 versus 77 days [p=0.02] and median 97 versus 141 days [p=0.004], respectively). Integrated transcriptomic and metabolomic analysis of ONC201-treated H3K27M-DMG cells revealed downregulation of TCA cycle, glycolysis, and pyruvate metabolism genes, most notably oxoglutarate dehydrogenase (OGDH), and concomitant changes in levels of associated metabolites including ɑ-ketoglutarate (ɑ-KG). Interestingly, L-2-hydroxyglutarate (L-2HG), a known inhibitor of the Jumonji C domain family of histone lysine demethylases, was also increased. This corresponded with increases in repressive H3K27me3 in vitro and in human tumors accompanied by epigenetic downregulation of cell cycle regulation and neuro-glial differentiation genes. Genetic knockdown of OGDH recapitulated ONC201-mediated increases in H3K27me3. In contrast, genetic knockdown of ClpP or lactate dehydrogenase A or overexpression of L-2-hydroxyglutarate dehydrogenase (L-2HGDH), but not D-2HGDH, abrogated ONC201-mediated increases in H3K27me3, demonstrating that ONC201 induces the production of L-2HG in H3K27M-DMG cells leading to increased H3K27me3. Overall, our data demonstrates the efficacy of ONC201 in H3K27M-mutant DMG and supports ONC201 as the first monotherapy to improve outcomes in patients with H3K27M-mutant DMG for whom few therapeutic options currently exist. Mechanistically, ONC201 disrupts integrated metabolic and epigenetic pathways and reverses pathognomonic H3K27me3 reduction.

The transsulfuration (TSS) pathway is an alternative source of cysteine for glutathione synthesis. Little of the TSS pathway in antioxidant capacity in sickle cell disease (SCD) is known. Here, we evaluate the effects of TSS pathway activation through cystathionine beta-synthase (CBS) to attenuate reactive oxygen species (ROS) and ferroptosis stresses in SCD. A vital contribution of the TSS pathway in sustaining cysteine levels is detected only under hemin exposure or physiological but not supraphysiological cystine supplement. Mechanistic studies show that hemin suppresses CBS expression to inhibit the TSS pathway and de novo cysteine biosynthesis. By contrast, the expression of CBS is inducible by dimethyl fumarate (DMF) through nuclear factor erythroid 2-related factor 2 (NRF2) activation and CpG islands DNA hydroxymethylation. DMF induces the expression of L-2-hydroxyglutarate dehydrogenase (L2HGDH) to downregulate L-2-hydroxyglutarate (L2HG) and increase global and locus-specific DNA hydroxymethylation levels. This DMF-upregulated DNA hydroxymethylation affects CBS locus chromatin structure modifications and upregulates gene expression. Our results suggest that CBS of the TSS pathway plays an important role in maintaining cysteine levels under restricted cystine availability or excess hemin exposure, and CBS upregulation by DMF increases the cellular glutathione levels to protect against ROS and ferroptosis stress in SCD.

Although clear cell renal cell carcinoma (ccRCC) has been shown to result in widespread aberrant cytosine methylation and loss of 5-hydroxymethylcytosine (5hmC), the prognostic impact and therapeutic targeting of this epigenetic aberrancy has not been fully explored. Analysis of 576 primary ccRCC samples demonstrated that loss of 5hmC was strongly associated with aggressive clinicopathologic features and was an independent adverse prognostic factor. Loss of 5hmC also predicted reduced progression-free survival after resection of nonmetastatic disease. The loss of 5hmC in ccRCC was not due to mutational or transcriptional inactivation of ten eleven translocation (TET) enzymes, but to their functional inactivation by l-2-hydroxyglutarate (L2HG), which was overexpressed due to the deletion and underexpression of L2HG dehydrogenase (L2HGDH). Ascorbic acid (AA) reduced methylation and restored genome-wide 5hmC levels via TET activation. Fluorescence quenching of the recombinant TET-2 protein was unaffected by L2HG in the presence of AA. Pharmacologic AA treatment led to reduced growth of ccRCC in vitro and reduced tumor growth in vivo, with increased intratumoral 5hmC. These data demonstrate that reduced 5hmC is associated with reduced survival in ccRCC and provide a preclinical rationale for exploring the therapeutic potential of high-dose AA in ccRCC.

A common single nucleotide polymorphism impairs B-cell activating factor receptor's multimerization, contributing to common variable immunodeficiency

Nrf2 sensitizes ferroptosis through l-2-hydroxyglutarate–mediated chromatin modifications in sickle cell disease