Richard Kirkman

l-2-Hydroxyglutarate: An Epigenetic Modifier and Putative Oncometabolite in Renal Cancer

UNLABELLED 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 that 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 2HG-mediated inhibition of ten-eleven translocation (TET) enzymes, which convert 5-methylcytosine (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. In addition, 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. SIGNIFICANCE Here, we report elevations of the putative oncometabolite l-2HG in the most common subtype of kidney cancer and describe a novel mechanism for the regulation of DNA 5hmC levels. Our findings provide new insight into the metabolic basis for the epigenetic landscape of renal cancer.

Developmental regulation of Th17-cell capacity in human neonates.

Human neonates are at significantly greater risk of serious infection than immunocompetent adults. In particular, very low birth weight infants in the neonatal intensive care nursery are at high risk of developing life‐threatening bacterial and fungal infections. Recent studies have identified Th17 cells as critical mediators of immunity to bacterial and fungal infections at epithelial barriers. Little is known, however, about the ontogeny of Th17‐cell responses in humans. The frequency of serious bacterial infections in preterm infants and the importance of Th17 cells in providing protection against such infections in animal studies prompted us to study Th17‐cell development in human neonates. Naïve CD4+ T cells from extremely preterm infants, term infants, and adults were assayed for their capacity to develop into Th17 effector cells. Surprisingly, Th17‐cell capacity was inversely related to developmental age. Neonates expressed higher levels of IL‐23R, RORγt, and STAT3 prior to activation and showed a significant Th17‐cell bias after activation. In contrast, adult cells expressed more TBX21 with a corresponding Th1‐cell bias. CD161 expression on Th17‐cell precursors was also developmentally regulated. Our results suggest there is significant developmental regulation of CD4+ effector lineages with a strong bias toward Th17‐cell development early in life.

Preferences for the selection of unique tRNA primers revealed from analysis of HIV-1 replication in peripheral blood mononuclear cells.

BackgroundAll human immunodeficiency virus (HIV-1) uses a host tRNALys,3 as the primer for reverse transcription. The tRNALys,3 is bound to a region on the HIV-1 genome, the primer-binding site (PBS), that is complementary to the 18 terminal nucleotides of tRNALys,3. How HIV-1 selects the tRNA from the intracellular milieu is unresolved.ResultsHIV-1 tRNA primer selection has been investigated using viruses in which the primer-binding site (PBS) and a sequence within U5 were altered so as to be complementary to tRNAMet, tRNAPro or tRNAIle. Analysis of the replication of these viruses in human peripheral blood mononuclear cells (PBMC) revealed preferences for the selection of certain tRNAs. HIV-1 with the PBS altered to be complementary to tRNAMet, with and without the additional mutation in U5 to be complementary to the anticodon of tRNAMet, stably maintains the PBS complementary to tRNAMet following extended in vitro culture in PBMC. In contrast, viruses with either the PBS or PBS and U5 mutated to be complementary to tRNAIle were unstable during in vitro replication in PBMC and reverted to utilize tRNALys,3. Viruses with the PBS altered to be complementary to tRNAPro replicated in PBMC but reverted to use tRNALys,3; viruses with mutations in both the U5 and PBS complementary to tRNAPro maintained this PBS, yet replicated poorly in PBMC.ConclusionThe results of these studies demonstrate that HIV-1 has preferences for selection of certain tRNAs for high-level replication in PBMC.

Retinoic Acid Hypersensitivity Promotes Peripheral Tolerance in Recent Thymic Emigrants

Whereas thymic education eliminates most self-reactive T cells, additional mechanisms to promote tolerance in the periphery are critical to prevent excessive immune responses against benign environmental Ags and some self-Ags. In this study we show that murine CD4+ recent thymic emigrants (RTEs) are programmed to facilitate tolerance in the periphery. Both in vitro and in vivo, naive RTEs more readily upregulate Foxp3 than do mature naive cells after stimulation under tolerogenic conditions. In RTEs, a relatively high sensitivity to retinoic acid contributes to decreased IFN-γ production, permitting the expression of Foxp3. Conversely, mature naive CD4 cells have a lower sensitivity to retinoic acid, resulting in increased IFN-γ production and subsequent IFN-γ–mediated silencing of Foxp3 expression. Enhanced retinoic acid signaling and Foxp3 induction in RTEs upon Ag encounter in the periphery may serve as form of secondary education that complements thymic education and helps avoid inappropriate immune responses. This mechanism for tolerance may be particularly important in settings where RTEs comprise a large fraction of the peripheral T cell pool, such as in newborns or after umbilical cord blood transplant.

tRNA isoacceptor preference prior to retrovirus gag-pol junction links primer selection and viral translation

ABSTRACT An essential step in the replication of all retroviruses is the capture of a cellular tRNA that is used as the primer for reverse transcription. The 3′-terminal 18 nucleotides of the tRNA are complementary to the primer binding site (PBS). Moloney murine leukemia virus (MuLV) preferentially captures tRNAPro. To investigate the specificity of primer selection, the PBS of MuLV was altered to be complementary to different tRNAs. Analysis of the infectivity of the virus and stability of the PBS following in vitro replication revealed that MuLV prefers to select tRNAPro, tRNAGly, or tRNAArg. Previous studies from our laboratory have suggested that tRNA primer capture is coordinated with translation. Coincidentally, a cluster of proline, arginine, and glycine precedes the Gag-Pol junction of MuLV. Human immunodeficiency virus type 1 (HIV-1), which prefers \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{3}^{\mathrm{Lys}}\) \end{document} as the primer, can be forced to utilize tRNAMet, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{1,2}^{\mathrm{Lys}}\) \end{document}, tRNAHis, or tRNAGlu, although these viruses replicate poorly. Codons for methionine, lysine, histidine, or glutamic acid are found prior to the Gag-Pol frameshift site. HIV-1 was mutated so that the 5 lysine codons prior to the Gag-Pol frameshift region were specific for \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{1,2}^{\mathrm{Lys}}\) \end{document}. HIV-1 forced to use \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{1,2}^{\mathrm{Lys}}\) \end{document} as the primer, with the mutation of codons specific for \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{1,2}^{\mathrm{Lys}}\) \end{document} prior to the Gag-Pol junction, had enhanced infectivity and replicated similarly to the wild-type virus. The results demonstrate that codon preference prior to the Gag-Pol junction influences primer selection and suggest a coordination of Gag-Pol synthesis and acquisition of the tRNA primer required for retrovirus replication.

Biochemical and Epigenetic Insights into L-2-Hydroxyglutarate, a Potential Therapeutic Target in Renal Cancer

Purpose: 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. Experimental Design: Genetic and pharmacologic approaches to modulate L-2-HG levels were assessed for effects on in vitro and in vivo 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. Results: L2HGDH suppresses both in vitro cell migration and in vivo tumor growth and these effects are mediated by L2HGDHs 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 in vitro phenotypes in an L-2-HG–dependent manner. Moreover, in vivo 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, L2HGDH copy loss, and lower L2HGDH expression are associated with tumor progression and/or worsened prognosis in patients with RCC. Conclusions: 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.

Abstract 4483: Functional implications ofPRDM16loss in kidney cancer

The most common genetic aberration associated with inherited or sporadic renal cell carcinoma (RCC) is the loss or nonfunctional mutations in the von Hippel-Lindau (VHL) tumor suppressor gene. The mutations lead to the stabilization of hypoxia inducible factor (HIF-1α and 2α), thereby promoting the expression of HIF target genes. HIF target genes are reported to induce tumor phenotypes by altering cellular metabolism and upregulating the expression of protumorigenic/angiogenic factors as well. We report here tissue microarray data and histology-based analyses demonstrating that the gene PRDM16 (PRD1-BF1-RIZ1 homologous domain containing 16) is silenced in many RCC tumors regardless of their VHL status. Bioinformatics analysis demonstrates a role for promoter methylation in silencing PRDM16. We investigated the role of PRDM16 in the context of VHL loss in kidney cancer. PRDM16 is known to regulate brown fat mitochondrial metabolism by inducing expressions of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PPARGC1A) and estrogen-related receptor-gamma (ESRRG). Altered cellular metabolism, in particular, decreased mitochondrial respiration is a hallmark of RCC, which is in attributed to VHL alterations. Analysis of TCGA data demonstrates that PRDM16 expression strongly correlates with PPARGC1A expression in RCC. Ectopic expression of PRDM16 in RCC cells can induce PPARGC-1α and ESRRG mRNA expression but fails to increase mitochondrial respiration. However, ectopic expression of PRDM16 in RCC cell lines can suppress both in vitro and in vivo tumor phenotypes. These data suggest that the tumor-suppressive activity of PRDM16 may be independent from its effects on mitochondrial metabolism. RNA-seq based analysis of a VHL mutated RCC line re-expressing PRDM16 demonstrates that a transcription suppressive role of PRDM16 predominates in RCC. PRDM16 suppresses genes involved in cell migration and proliferation. In particular, PRDM16 downregulates the expression of semaphorin 5B (SEMA5B). q-PCR analysis validates high-throughput data in a panel of RCC and embryonic kidney cell lines. SEMA5B knock down significantly decreases RCC proliferation supporting the protumorigenic function of SEMA5B in RCC. Given the central role of VHL loss in renal tumor initiation, we demonstrate that the VHL/HIF axis regulates the expression of SEMA5B in RCC. Collectively, these data suggest that transcription suppressive activity of PRDM16 and VHL controls expression of the protumorigenic factor SEMA5B and that epigenetic silencing of PRDM16 thus amplifies this consequence of VHL loss. Citation Format: Anirban Kundu, Eun-Young Kho, Sandeep B. Shelar, Hyeyoung Nam, Garret Brinkley, Shimoga Darshan, Yawen Tang, Richard Kirkman, David K. Crossman, Sooryanarayana Varambally, Glenn C. Rowe, Shi Wei, Phillip Buckhaults, Sunil Sudarshan. Functional implications of PRDM16 loss in 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 4483.

Abstract LB-131: Elevated (L) -2-hydroxyglutarate promotes loss of 5-hydroxymethylcytosine in clear cell renal cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA We undertook an unbiased metabolomics profile of clear cell renal cell carcinoma (ccRCC), the most common histological subtype, to gain deeper insight into the metabolic basis of this disease. Here, we demonstrate elevations of the metabolite 2-hydroxyglutarate (2HG) in kidney cancer. Elevation of 2HG, specifically the D- enantiomer, has been linked to transformation and identified in the context of gain of function mutations of isocitrate dehydrogenase (IDH1 and IDH2). Notably, enantiomeric resolution demonstrates that, in contrast to IDH1/IDH2 mutant tumors, kidney tumors demonstrate elevations of the L enantiomer of 2HG (L-2HG). Moreover, high 2HG tumors demonstrate reduced DNA levels of 5-hydroxymethylcytosine (5hmC) consistent with the ability of L-2HG to inhibit TET (Ten Eleven Translocation) enzymes which convert 5-methylcystoine to 5-hmC. Finally, we demonstrate that these changes are mediated by the reduced expression of L-2HG dehydrogenase (L2HGDH) in ccRCC. Here, we report elevations of the putative oncometabolite L-2HG in the most common subtype of kidney cancer and describe a novel mechanism for the regulation of DNA 5hmC levels. Our findings provide new insight into the metabolic basis for the epigenetic landscape of renal cancer. Citation Format: Eun-Hee Shim, Carolina B. Livi, John Knight, Ross P. Holmes, Dinesh Rakheja, Sadanan Velu, Eun-Young Kho, Balachandra Chenna, Shane L. Rea, Daniel Benson, Richard Kirkman, Arindam Ghosh, Qiuhua Li, Sejong Bae, Shi Wei, Karen L. Block, Sunil Sudarshan. Elevated (L) -2-hydroxyglutarate promotes loss of 5-hydroxymethylcytosine in clear cell renal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-131. doi:10.1158/1538-7445.AM2014-LB-131

Abstract 2435: Novel regulators of renal cancer metabolism

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Renal cell carcinoma (RCC) is among the 10 most common malignancies in the U.S. Clear cell RCC (ccRCC) is the most common histology. Recent analysis of ccRCC by the Cancer Genome Atlas (TCGA) demonstrates the reduced mRNA expression of several enzymes of the tricarboxylic acid (TCA) cycle. Moreover, reduced TCA enzymes expression is associated with worse prognosis. These data indicate a transcriptional program that promotes a bioenergetic shift. These studies prompted us to examine transcription factors that are known regulators of mitochondrial metabolism including the estrogen-related receptors (ERRs) and peroxisome proliferator-activated receptor γ (PPARγ) coactivators 1 (PGC-1s). Interestingly, mRNA analysis of RCC tissues and cell lines demonstrates the reduced expression of ERRγ and PGC-1α. Notably, prior analysis demonstrates putative ERR binding sites in the promoters of FH and MDH2, two genes encoding TCA enzymes with reduced expression in ccRCC. Promoter analysis of these genes with a luciferase reporter assay demonstrates that ERRγ and PGC-1α cooperate to regulate the expression of TCA enzymes indicating that reduced expression of these factors promotes reduced TCA enzyme expression in RCC. Citation Format: EunYoung Kho, Richard Kirkman, Eun-Hee Shim, Arindam Goash, Sunil Sudarshan. Novel regulators of renal cancer metabolism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2435. doi:10.1158/1538-7445.AM2014-2435