L-2-hydroxyglutarate remodeling of the epigenome and epitranscriptome creates a metabolic vulnerability in kidney cancer models.

Tumor cells are known to undergo considerable metabolic reprogramming to meet their unique demands and drive tumor growth. At the same time, this reprogramming may come at a cost with resultant metabolic vulnerabilities. The small molecule L-2-hdroxyglutarate (L-2HG) is elevated in the most common histology of renal cancer. Similar to other oncometabolites, L-2HG has the potential to profoundly impact gene expression. Here, we demonstrate that L-2HG remodels amino acid metabolism in renal cancer cells through the combined effects on histone methylation and RNA N6-methyladenosine (m6A). The combined effects of L-2HG result in a metabolic liability that renders tumors cells reliant on exogenous serine to support proliferation, redox homeostasis, and tumor growth. In concert with these data, high L-2HG kidney cancers demonstrates reduced expression of multiple serine biosynthetic enzymes. Collectively, our data indicate that high L-2HG renal tumors could be specifically targeted by strategies that limit serine availability to tumors.

Whole-Exome and Transcriptome Analysis of UV-Exposed Epidermis and Carcinoma In Situ Reveals Early Drivers of Carcinogenesis.

Squamous cell carcinoma in situ (SCCIS) is a prevalent precancerous lesion that can progress to cutaneous squamous cell carcinoma. Although SCCIS is common, its pathogenesis remains poorly understood. To better understand SCCIS development, we performed laser captured microdissection of human SCCIS and the adjacent epidermis to isolate genomic DNA and RNA for next-generation sequencing. Whole-exome sequencing identified UV-signature mutations in multiple genes, including NOTCH1-3 in the epidermis and SCCIS and oncogenic TP53 mutations in SCCIS. Gene families, including SLFN genes, contained UV/oxidative-signature disruptive epidermal mutations that manifested positive selection in SCCIS. The frequency and distribution of NOTCH and TP53 mutations indicate that NOTCH mutations may precede TP53 mutations. RNA sequencing identified 1,166 differentially expressed genes; the top five enriched gene ontology biological processes included (i) immune response, (ii) epidermal development, (iii) protein phosphorylation, (iv) regulation of catalytic activity, and (v) cytoskeletal regulation. The NEURL1 ubiquitin ligase, which targets Notch ligands for degradation, was upregulated in SCCIS. NEURL1 protein was found to be elevated in SCCIS suggesting that increased levels could represent a mechanism for downregulating Notch during UV-induced carcinogenesis. The data from DNA and RNA sequencing of epidermis and SCCIS provide insights regarding SCCIS formation.

Teleological role of L-2-hydroxyglutarate dehydrogenase in the kidney.

L-2-hydroxyglutarate (L-2HG) is an oncometabolite found elevated in renal tumors. However, this molecule might have physiological roles that extend beyond its association with cancer, as L-2HG levels are elevated in response to hypoxia and during Drosophila larval development. L-2HG is known to be metabolized by L-2HG dehydrogenase (L2HGDH), and loss of L2HGDH leads to elevated L-2HG levels. Despite L2HGDH being highly expressed in the kidney, its role in renal metabolism has not been explored. Here, we report our findings utilizing a novel CRISPR/Cas9 murine knockout model, with a specific focus on the role of L2HGDH in the kidney. Histologically, L2hgdh knockout kidneys have no demonstrable histologic abnormalities. However, GC-MS metabolomics demonstrates significantly reduced levels of the TCA cycle intermediate succinate in multiple tissues. Isotope labeling studies with [U-13C] glucose demonstrate that restoration of L2HGDH in renal cancer cells (which lowers L-2HG) leads to enhanced incorporation of label into TCA cycle intermediates. Subsequent biochemical studies demonstrate that L-2HG can inhibit the TCA cycle enzyme α-ketoglutarate dehydrogenase. Bioinformatic analysis of mRNA expression data from renal tumors demonstrates that L2HGDH is co-expressed with genes encoding TCA cycle enzymes as well as the gene encoding the transcription factor PGC-1α, which is known to regulate mitochondrial metabolism. Restoration of PGC-1α in renal tumor cells results in increased L2HGDH expression with a concomitant reduction in L-2HG levels. Collectively, our analyses provide new insight into the physiological role of L2HGDH as well as mechanisms that promote L-2HG accumulation in disease states.

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 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 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.

Primary Cutaneous T-cell Lymphoma With Coexpression of T-Cell Receptors αß and γδ.

T lymphocytes belong to 2 distinct sublineages that express either αß or γδ T-cell receptor (TCR) complex. Although malignancy is a great instigator of lineage infidelity, as exemplified by aberrant expression of numerous lineage markers in lymphoma cells, malignant T cells rarely coexpress αß and γδ TCR complexes. Similarly, only rare cases of CD4/CD8 double-positive primary cutaneous T-cell lymphoma have been reported. In this report, we describe a remarkable case of primary cutaneous T-cell lymphoma coexpressing αß and γδ TCR complexes, strong diffuse CD8, and a very restricted coexpression of CD4 and CD8. A 66-year-old man was referred to our center for treatment of a persistent eczematoid eruption of 6 years of duration. An initial biopsy demonstrated not only marked spongiosis, but also an epidermotropic population of CD4 small mature T cells with partial expression of CD8. The process remained indolent for another year, followed by an abrupt progression with development of plaques and tumors. Repeat biopsies of these lesions demonstrated a superimposed population of large anaplastic T cells extensively involving the dermis and epidermis. The large cells showed a strong uniform expression of CD3, CD8, CD45RA, CD5, granzyme, TIA1, perforin, TCR-ß, and TCR-γ and a weaker but unambiguous expression of CD4, CD25, CD2, and CD56. TCR gene rearrangement studies showed clonal rearrangements for TCR-ß and TCR-γ with identical peaks to those seen in the biopsy from a year earlier. The patient developed lymphadenopathy, with a biopsy showing nodal involvement by a morphologically and phenotypically identical neoplastic T-cell population. The disease showed partial response to systemic chemotherapy with development of new plaques, but these new lesions have regressed with radiation therapy.

Point mutations of the mTOR-RHEB pathway in renal cell carcinoma.

Aberrations in the mTOR (mechanistic target of rapamycin) axis are frequently reported in cancer. Using publicly available tumor genome sequencing data, we identified several point mutations in MTOR and its upstream regulator RHEB (Ras homolog enriched in brain) in patients with clear cell renal cell carcinoma (ccRCC), the most common histology of kidney cancer. Interestingly, we found a prominent cluster of hyperactivating mutations in the FAT (FRAP-ATM-TTRAP) domain of mTOR in renal cell carcinoma that led to an increase in both mTORC1 and mTORC2 activities and led to an increased proliferation of cells. Several of the FAT domain mutants demonstrated a decreased binding of DEPTOR (DEP domain containing mTOR-interacting protein), while a subset of these mutations showed altered binding of the negative regulator PRAS40 (proline rich AKT substrate 40). We also identified a recurrent mutation in RHEB in ccRCC patients that leads to an increase in mTORC1 activity. In vitro characterization of this RHEB mutation revealed that this mutant showed considerable resistance to TSC2 (Tuberous Sclerosis 2) GAP (GTPase activating protein) activity, though its interaction with TSC2 remained unaltered. Mutations in the FAT domain of MTOR and in RHEB remained sensitive to rapamycin, though several of these mutations demonstrated residual mTOR kinase activity after treatment with rapamycin at clinically relevant doses. Overall, our data suggests that point mutations in the mTOR pathway may lead to downstream mTOR hyperactivation through multiple different mechanisms to confer a proliferative advantage to a tumor cell.

Another small molecule in the oncometabolite mix: L-2-Hydroxyglutarate in kidney cancer.

Alterations in metabolism are now considered a hallmark of cancer. One of the clearest links between metabolism and malignancy are oncometabolites. To date, several putative oncometabolites with transforming properties have been identified in the context of tumors due to both gain and loss of function mutations in genes encoding enzymes of intermediary metabolism. Through an unbiased metabolomics approach, we identified elevations of the metabolite 2-hydroxyglutarate (2-HG) in the most common histology of kidney cancer that is among the most common malignancies in both men and women. Subsequent analyses demonstrate that the predominant enantiomer of 2-HG elevated in renal cancer is the L(S) form. Notably, elevations of L-2HG are due in part to loss of expression of the L-2HG dehydrogenase (L2HGDH) which normally serves as an enzyme of "metabolite repair" to keep levels of this metabolite from accumulating. Lowering L-2HG levels in RCC through re-expression of L2HGDH mitigates tumor phenotypes and reverses epigenetic alterations known to be targeted by oncometabolites. These data add to the growing body of evidence that metabolites, similarly to oncogenes and oncoproteins, can play a role in tumor development and/or progression. As such, they represent a unique opportunity to utilize these findings in the clinic setting.

The Association of Lung Function, Bronchial Hyperresponsiveness, and Exhaled Nitric Oxide Differs Between Atopic and Non-atopic Asthma in Children.

PURPOSE: Although many previous studies have attempted to identify differences between atopic asthma (AA) and non-atopic asthma (NAA), they have mainly focused on the difference of each variable of lung function and airway inflammation. The aim of this study was to evaluate relationships between lung function, bronchial hyperresponsiveness (BHR), and the exhaled nitric oxide (eNO) levels in children with AA and NAA. METHODS: One hundred and thirty six asthmatic children aged 5-15 years and 40 normal controls were recruited. Asthma cases were classified as AA (n=100) or NAA (n=36) from skin prick test results. Lung function, BHR to methacholine and adenosine-5'-monophosphate (AMP), eNO, blood eosinophils, and serum total IgE were measured. RESULTS: The AA and NAA cases shared common features including a reduced small airway function and increased BHR to methacholine. However, children with AA showed higher BHR to AMP and eNO levels than those with NAA. When the relationships among these variables in the AA and NAA cases were evaluated, the AA group showed significant relationships between lung function, BHR to AMP or methacholine and eNO levels. However, the children in the NAA group showed an association between small airway function and BHR to methacholine only. CONCLUSIONS: These findings suggest that the pathogenesis of NAA may differ from that of AA during childhood in terms of the relationship between lung function, airway inflammation and BHR.

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.

The oncometabolite fumarate promotes pseudohypoxia through noncanonical activation of NF-κB signaling.

Inactivating mutations of the gene encoding the tricarboxylic acid cycle enzyme fumarate hydratase (FH) have been linked to an aggressive variant of hereditary kidney cancer (hereditary leiomyomatosis and renal cell cancer). These tumors accumulate markedly elevated levels of fumarate. Fumarate is among a growing list of oncometabolites identified in cancers with mutations of genes involved in intermediary metabolism. FH-deficient tumors are notable for their pronounced accumulation of the transcription factor hypoxia inducible factor-1α (HIF-1α) and aggressive behavior. To date, HIF-1α accumulation in hereditary leiomyomatosis and renal cell cancer tumors is thought to result from fumarate-dependent inhibition of prolyl hydroxylases and subsequent evasion from von Hippel-Lindau-dependent degradation. Here, we demonstrate a novel mechanism by which fumarate promotes HIF-1α mRNA and protein accumulation independent of the von Hippel-Lindau pathway. Here we demonstrate that fumarate promotes p65 phosphorylation and p65 accumulation at the HIF-1α promoter through non-canonical signaling via the upstream Tank binding kinase 1 (TBK1). Consistent with these data, inhibition of the TBK1/p65 axis blocks HIF-1α accumulation in cellular models of FH loss and markedly reduces cell invasion of FH-deficient RCC cancer cells. Collectively, our data demonstrate a novel mechanism by which pseudohypoxia is promoted in FH-deficient tumors and identifies TBK1 as a novel putative therapeutic target for the treatment of aggressive fumarate-driven tumors.

The p53-induced factor Ei24 inhibits nuclear import through an importin ß-binding-like domain.

The etoposide-induced protein Ei24 was initially identified as a p53-responsive, proapoptotic factor, but no clear function has been described. Here, we use a nonbiased proteomics approach to identify members of the importin (IMP) family of nuclear transporters as interactors of Ei24 and characterize an IMPß-binding-like (IBBL) domain within Ei24. We show that Ei24 can bind specifically to IMPß1 and IMPα2, but not other IMPs, and use a mutated IMPß1 derivative to show that Ei24 binds to the same site on IMPß1 as the IMPα IBB. Ectopic expression of Ei24 reduced the extent of IMPß1- or IMPα/ß1-dependent nuclear protein import specifically, whereas specific alanine substitutions within the IBBL abrogated this activity. Induction of endogenous Ei24 expression through etoposide treatment similarly inhibited nuclear import in a mouse embryonic fibroblast model. Thus, Ei24 can bind specifically to IMPß1 and IMPα2 to impede their normal role in nuclear import, shedding new light on the cellular functions of Ei24 and its tumor suppressor role.

Regulation of delta-aminolevulinic acid dehydratase by krüppel-like factor 1.

Krüppel-like factor 1(KLF1) is a hematopoietic-specific zinc finger transcription factor essential for erythroid gene expression. In concert with the transacting factor GATA1, KLF1 modulates the coordinate expression of the genes encoding the multi-enzyme heme biosynthetic pathway during erythroid differentiation. To explore the mechanisms underpinning KLF1 action at the gene loci regulating the first 3 steps in this process, we have exploited the K1-ERp erythroid cell line, in which KLF1 translocates rapidly to the nucleus in response to treatment with 4-OH-Tamoxifen (4-OHT). KLF1 acts as a differentiation-independent transcriptional co-regulator of delta-aminolevulinic acid dehydratase (Alad), but not 5-aminolevulinate synthase gene (Alas2) or porphobilinogen deaminase (Pbgd). Similar to its role at the ß-globin promoter, KLF1 induces factor recruitment and chromatin changes at the Alad1b promoter in a temporally-specific manner. In contrast to these changes, we observed a distinct mechanism of histone eviction at the Alad1b promoter. Furthermore, KLF1-dependent events were not modulated by GATA1 factor promoter co-occupancy alone. These results not only enhance our understanding of erythroid-specific modulation of heme biosynthetic regulation by KLF1, but provide a model that will facilitate the elucidation of novel KLF1-dependent events at erythroid gene loci that are independent of GATA1 activity.

Expression of the F-box protein SKP2 induces hyperplasia, dysplasia, and low-grade carcinoma in the mouse prostate.

Low or absent expression of the cyclin-dependent kinase inhibitor p27(Kip1) serves as an excellent malignant marker for prostate and other human cancers. The level of p27(Kip1) is regulated primarily by the ubiquitin E3 ligase SCF(SKP2) through ubiquitin-dependent proteolysis. Expression of the F-box protein SKP2 is inversely correlated with p27 in many cancers. To determine the role of SCF(SKP2) in proliferation and tumorigenesis, we established transgenic mouse lines that specifically expressed SKP2 in the prostate gland. Unscheduled expression of SKP2 promoted marked overproliferation, resulting in hyperplasia, dysplasia, and low-grade carcinoma in the prostate gland. Consistent with its critical role in p27 proteolysis, SKP2 expression caused significant down-regulation of p27 in prostate glands from transgenic animals. Immunohistological staining indicated that SKP2 expression is restricted to the hyperplastic/dysplastic regions and that there is an inverse relationship between SKP2 and p27 expression in the ductal epithelium in transgenic animals. The prostate glands from transgenic mice also exhibited high levels of proliferative and mitotic markers such as Ki67 and cyclin B1. Our data suggest that SKP2 acts as an oncoprotein in the mouse prostate gland, probably through its function as a limiting factor for ubiquitin-dependent degradation of p27.

Targeted disruption of hsp70.1 sensitizes to osmotic stress.

The 70 kDa heat shock protein (Hsp70) plays a critical role in cell survival and thermotolerance in response to various stress stimuli. Two nearly identical genes, hsp70.1 and hsp70.3, in response to environmental stress, rapidly induce Hsp70. However, it remains unclear whether these two genes are differentially regulated by various stresses. To address the physiological role of the hsp70.1 and hsp70.3 genes in the stress response, we generated mice that specifically lack hsp70.1. In contrast to heat shock, which rapidly induced both hsp70.1 and hsp70.3 mRNA, osmotic stress selectively induced transcription of hsp70.1. In hsp70.1-deficient embryonic fibroblasts, osmotic stress markedly reduced cell viability. Furthermore, when osmotic stress was applied in vivo, hsp70.1-deficient mice exhibited increased apoptosis in the renal medulla. Taken together, our results demonstrate that differential expression of hsp70 genes contributes to the stress response and that the hsp70.1 gene plays a critical role in osmotolerance.