fumarates and Leiomyomatosis

Biallelic inactivation of fumarate hydratase(FH) causes type 2 papillary renal cell carcinoma (PRCC2), uterine fibroids, and cutaneous leimyomas, a condition known as hereditary leiomyomatosis and renal cell cancer(HLRCC). The most direct effect of FH inactivation is intracellular fumarate accumulation. A majority of studies on FH inactivation over the past decade have focused on the theory that intracellular fumarate stabilizes hypoxia-inducible factor 1α(HIF1A) through competitive inhibition of HIF prolyl hydroxylases. Recently, a competing theory that intracellular fumarate activates nuclear factor (erythroid-derived 2)-like 2(NRF2) through post-translational modification of its negative regulator. Kelch-like ECH-associated protein 1(KEAP1) has emerged from a computational modeling study and mouse model studies. This review dissects the origin of these two governing theories and highlights the presence of chromatin-structure-regulated targets of transcription factors, which we refer to as "cryptic targets" of transcription factors. One such cryptic target is heme oxygenase I(HMOX1), the expression of which is known to be modulated by the gene product of SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 (SMARCA4, also known as BRG1).

Topics: Animals; DNA Helicases; Fumarate Hydratase; Fumarates; Heme Oxygenase-1; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Intracellular Signaling Peptides and Proteins; Kelch-Like ECH-Associated Protein 1; Kidney Neoplasms; Leiomyomatosis; Neoplastic Syndromes, Hereditary; NF-E2-Related Factor 2; Nuclear Proteins; Procollagen-Proline Dioxygenase; Protein Processing, Post-Translational; Skin Neoplasms; Transcription Factors; Uterine Neoplasms

: Hereditary leiomyomatosis (HL) is an autosomal dominant condition due to a variety of fumarate hydratase (FH) mutations in which individuals tend to develop cutaneous leiomyomas, multiple uterine leiomyomas and are at risk for developing aggressive papillary renal cell carcinoma.. : A 26-year-old man with a past history of acute lymphoblastic leukemia (T-ALL) presented with numerous painful light brown papules and nodules spread all over his body except for the head, appearing since infancy. Similar lesions were present in his mother's family. A cutaneous biopsy revealed a cutaneous leiomyoma. His mother died from metastatic uterine neoplasia and his sister suffered from leiomyoma of the uterus. No renal cancer was reported in his family. A heterozygous pathogenic variant was detected in the FH gene.. : To our knowledge, this is the first case possibly linking HL and T-ALL through FH deficiency.

Topics: Adult; Carcinoma, Renal Cell; Female; Fumarates; Genetic Predisposition to Disease; Humans; Kidney Neoplasms; Leiomyomatosis; Male; Mutation; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Skin Neoplasms; Uterine Neoplasms

The

Topics: Adult; Autophagy; Base Sequence; Female; Fumarate Hydratase; Fumarates; HEK293 Cells; Humans; Leiomyomatosis; Male; Mutation; Pedigree; Protein Multimerization; Signal Transduction; TOR Serine-Threonine Kinases; Uterine Neoplasms

Background Noninvasive in vivo detection of fumarate accumulation may help identify fumarate hydratase deficiency in renal cancer related to hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome. Purpose To investigate the feasibility of MR spectroscopy (MRS) in detecting elevated fumarate levels in HLRCC-associated renal cancers. Materials and Methods This study included an experimental xenograft mouse model and prospective clinical cohort. First, MRS was performed on patient-derived tumor xenograft models and control models to detect fumarate. Then, consecutive participants with clinical suspicion of HLRCC-associated renal tumors were enrolled. For the detection of fumarate, MRS results were classified as detected, borderline, undetected, or technical failure. The sensitivity, specificity, and accuracy of MRS for diagnosing HLRCC-associated renal cancer were assessed. The signal-to-noise ratio (SNR) of the fumarate peak was calculated and evaluated with receiver operating characteristic curve analysis. Results Fumarate peaks were detected at 6.54 parts per million in all three patient-derived xenograft models. A total of 38 participants (21 men; mean age, 47 years [range, 18-71 years]) with 46 lesions were analyzed. All primary HLRCC-associated renal cancers showed a fumarate peak; among the seven metastatic HLRCC-associated lesions, a fumarate peak was detected in three lesions and borderline in two. When only detected peaks were regarded as positive findings, the sensitivity, specificity, and accuracy of MRS at the lesion level were 69% (nine of 13 lesions), 100% (33 of 33 lesions), and 91% (42 of 46 lesions), respectively. When borderline peaks were also included as a positive finding, the sensitivity, specificity, and accuracy reached 85% (11 of 13 lesions), 88% (29 of 33 lesions), and 87% (40 of 46 lesions), respectively. The SNR of fumarate showed an area under the receiver operating characteristic curve of 0.87 for classifying HLRCC-associated tumors. Conclusion MR spectroscopy of fumarate was sensitive and specific for hereditary leiomyomatosis and renal cell carcinoma-associated tumors. © RSNA, 2022

Topics: Animals; Carcinoma, Renal Cell; Female; Fumarates; Humans; Kidney Neoplasms; Leiomyomatosis; Magnetic Resonance Spectroscopy; Mice; Neoplastic Syndromes, Hereditary; Prospective Studies; Skin Neoplasms; Syndrome; Uterine Neoplasms

Inherited pathogenic variants in genes encoding the metabolic enzymes succinate dehydrogenase (SDH) and fumarate hydratase predispose to tumor development through accumulation of oncometabolites (succinate and fumarate, respectively; ref. 1). Noninvasive. Magnetic resonance spectroscopy (. Here, we have demonstrated a proof of principle that. This study demonstrates that

Topics: Adult; Female; Fumarate Hydratase; Fumarates; Germ-Line Mutation; Humans; Kidney Neoplasms; Leiomyomatosis; Male; Middle Aged; Neoplastic Syndromes, Hereditary; Proton Magnetic Resonance Spectroscopy; Skin Neoplasms; Succinate Dehydrogenase; Uterine Neoplasms

An important context in which metabolism influences tumorigenesis is the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a disease in which mutation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) causes hyperaccumulation of fumarate. This electrophilic oncometabolite can alter gene activity at the level of transcription, via reversible inhibition of epigenetic dioxygenases, as well as posttranslationally, via covalent modification of cysteine residues. To better understand the potential for metabolites to influence posttranslational modifications important to tumorigenesis and cancer cell growth, here we report a chemoproteomic analysis of a kidney-derived HLRCC cell line. Using a general reactivity probe, we generated a data set of proteomic cysteine residues sensitive to the reduction in fumarate levels caused by genetic reintroduction of active FH into HLRCC cell lines. This revealed a broad up-regulation of cysteine reactivity upon FH rescue, which evidence suggests is caused by an approximately equal proportion of transcriptional and posttranslational modification-mediated regulation. Gene ontology analysis highlighted several new targets and pathways potentially modulated by FH mutation. Comparison of the new data set with prior studies highlights considerable heterogeneity in the adaptive response of cysteine-containing proteins in different models of HLRCC. This is consistent with emerging studies indicating the existence of cell- and tissue-specific cysteine-omes, further emphasizing the need for characterization of diverse models. Our analysis provides a resource for understanding the proteomic adaptation to fumarate accumulation and a foundation for future efforts to exploit this knowledge for cancer therapy.

Topics: Cell Line, Tumor; Cysteine; Fumarate Hydratase; Fumarates; Humans; Kidney Neoplasms; Leiomyomatosis; Neoplastic Syndromes, Hereditary; Skin Neoplasms; Uterine Neoplasms

Hereditary cancer disorders often provide an important window into novel mechanisms supporting tumor growth. Understanding these mechanisms thus represents a vital goal. Toward this goal, here we report a chemoproteomic map of fumarate, a covalent oncometabolite whose accumulation marks the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC). We applied a fumarate-competitive chemoproteomic probe in concert with LC-MS/MS to discover new cysteines sensitive to fumarate hydratase (FH) mutation in HLRCC cell models. Analysis of this dataset revealed an unexpected influence of local environment and pH on fumarate reactivity, and enabled the characterization of a novel FH-regulated cysteine residue that lies at a key protein-protein interface in the SWI-SNF tumor-suppressor complex. Our studies provide a powerful resource for understanding the covalent imprint of fumarate on the proteome and lay the foundation for future efforts to exploit this distinct aspect of oncometabolism for cancer diagnosis and therapy.

Topics: Cell Line, Tumor; Chromatography, Liquid; Cysteine; Fumarates; HEK293 Cells; Humans; Hydrogen-Ion Concentration; Leiomyomatosis; Models, Biological; Neoplastic Syndromes, Hereditary; Proteomics; Signal Transduction; Skin Neoplasms; Tandem Mass Spectrometry; Uterine Neoplasms

Dysregulated cellular metabolism is an emerging hallmark of cancer. Improved methods to profile aberrant metabolic activity thus have substantial applications as tools for diagnosis and understanding the biology of malignant tumors. Here we describe the utilization of a bioorthogonal ligation to fluorescently detect the TCA cycle oncometabolite fumarate. This method enables the facile measurement of fumarate hydratase activity in cell and tissue samples, and can be used to detect disruptions in metabolism that underlie the genetic cancer syndrome hereditary leiomyomatosis and renal cell cancer (HLRCC). The current method has substantial utility for sensitive fumarate hydratase activity profiling, and also provides a foundation for future applications in diagnostic detection and imaging of cancer metabolism.

Topics: Citric Acid Cycle; Click Chemistry; Cycloaddition Reaction; Enzyme Assays; Female; Fluorescent Dyes; Fluorometry; Fumarate Hydratase; Fumarates; Humans; Leiomyomatosis; Neoplastic Syndromes, Hereditary; Skin Neoplasms; Uterine Neoplasms

Topics: Adrenal Gland Neoplasms; Cell Line; Cell Line, Tumor; Citric Acid Cycle; DNA Breaks, Double-Stranded; Fumarates; Germ-Line Mutation; HEK293 Cells; Humans; Leiomyomatosis; Neoplastic Syndromes, Hereditary; Pheochromocytoma; Recombinational DNA Repair; Skin Neoplasms; Succinic Acid; Uterine Neoplasms

Germ line mutations of the gene encoding the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (

Topics: Amino Acid Sequence; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Ferritins; Forkhead Box Protein M1; Fumarate Hydratase; Fumarates; Humans; Intracellular Space; Iron Regulatory Protein 2; Kidney Neoplasms; Leiomyomatosis; Models, Biological; NF-E2-Related Factor 2; Protein Biosynthesis; Signal Transduction; Succinic Acid; Transcription, Genetic

The gene encoding the Krebs cycle enzyme fumarate hydratase (FH) is mutated in hereditary leiomyomatosis and renal cell cancer (HLRCC). Loss of FH activity causes accumulation of intracellular fumarate, which can directly modify cysteine residues to form 2-succinocysteine through succination. We undertook a proteomic-based screen in cells and renal cysts from Fh1 (murine FH)-deficient mice and identified 94 protein succination targets. Notably, we identified the succination of three cysteine residues in mitochondrial Aconitase2 (ACO2) crucial for iron-sulfur cluster binding. We show that fumarate exerts a dose-dependent inhibition of ACO2 activity, which correlates with increased succination as determined by mass spectrometry, possibly by interfering with iron chelation. Importantly, we show that aconitase activity is impaired in FH-deficient cells. Our data provide evidence that succination, resulting from FH deficiency, targets and potentially alters the function of multiple proteins and may contribute to the dysregulated metabolism observed in HLRCC.

Topics: Aconitate Hydratase; Animals; Cell Line; Cysteine; Fumarate Hydratase; Fumarates; Humans; Iron; Kidney Neoplasms; Leiomyomatosis; Mice; Mice, Transgenic; Mitochondria; Neoplastic Syndromes, Hereditary; Proteome; Skin Neoplasms; Succinic Acid; Uterine Neoplasms

Loss-of-function mutations of the tumor suppressor gene encoding fumarase (FH) occur in individuals with hereditary leiomyomatosis and renal cell cancer syndrome (HLRCC). We found that loss of FH activity conferred protection from apoptosis in normal human renal cells and fibroblasts. In FH-defective cells, both hypoxia-inducible factor 1α (HIF-1α) and HIF-2α accumulated, but they were not required for apoptosis protection. Conversely, AMP-activated protein kinase (AMPK) was activated and required, as evidenced by the finding that FH inactivation failed to protect AMPK-null mouse embryo fibroblasts (MEFs) and AMPK-depleted human renal cells. Activated AMPK was detected in renal cysts, which occur in mice with kidney-targeted deletion of Fh1 and in kidney cancers of HLRCC patients. In Fh1-null MEFs, AMPK activation was sustained by fumarate accumulation and not by defective energy metabolism. Addition of fumarate and succinate to kidney cells led to extracellular signal-regulated kinase 1/2 (ERK1/2) and AMPK activation, probably through a receptor-mediated mechanism. These findings reveal a new mechanism of tumorigenesis due to FH loss and an unexpected pro-oncogenic role for AMPK that is important in considering AMPK reactivation as a therapeutic strategy against cancer.

Topics: AMP-Activated Protein Kinases; Animals; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Cell Line; Fumarate Hydratase; Fumarates; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney Neoplasms; Leiomyomatosis; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplastic Syndromes, Hereditary; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering; Signal Transduction; Skin Neoplasms; Tumor Suppressor Proteins; Uterine Neoplasms

Fumarate hydratase (FH) is an enzyme of the tricarboxylic acid cycle (TCA cycle) that catalyses the hydration of fumarate into malate. Germline mutations of FH are responsible for hereditary leiomyomatosis and renal-cell cancer (HLRCC). It has previously been demonstrated that the absence of FH leads to the accumulation of fumarate, which activates hypoxia-inducible factors (HIFs) at normal oxygen tensions. However, so far no mechanism that explains the ability of cells to survive without a functional TCA cycle has been provided. Here we use newly characterized genetically modified kidney mouse cells in which Fh1 has been deleted, and apply a newly developed computer model of the metabolism of these cells to predict and experimentally validate a linear metabolic pathway beginning with glutamine uptake and ending with bilirubin excretion from Fh1-deficient cells. This pathway, which involves the biosynthesis and degradation of haem, enables Fh1-deficient cells to use the accumulated TCA cycle metabolites and permits partial mitochondrial NADH production. We predicted and confirmed that targeting this pathway would render Fh1-deficient cells non-viable, while sparing wild-type Fh1-containing cells. This work goes beyond identifying a metabolic pathway that is induced in Fh1-deficient cells to demonstrate that inhibition of haem oxygenation is synthetically lethal when combined with Fh1 deficiency, providing a new potential target for treating HLRCC patients.

Topics: Animals; Bilirubin; Cell Line; Cells, Cultured; Citric Acid Cycle; Computer Simulation; Fumarate Hydratase; Fumarates; Genes, Lethal; Genes, Tumor Suppressor; Glutamine; Heme; Heme Oxygenase (Decyclizing); Kidney Neoplasms; Leiomyomatosis; Mice; Mitochondria; Mutation; NAD; Neoplastic Syndromes, Hereditary; Skin Neoplasms; Uterine Neoplasms

In eukaryotes, fumarase (FH in human) is a well-known tricarboxylic-acid-cycle enzyme in the mitochondrial matrix. However, conserved from yeast to humans is a cytosolic isoenzyme of fumarase whose function in this compartment remains obscure. A few years ago, FH was surprisingly shown to underlie a tumor susceptibility syndrome, Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC). A biallelic inactivation of FH has been detected in almost all HLRCC tumors, and therefore FH was suggested to function as a tumor suppressor. Recently it was suggested that FH inhibition leads to elevated intracellular fumarate, which in turn acts as a competitive inhibitor of HPH (HIF prolyl hydroxylase), thereby causing stabilization of HIF (Hypoxia-inducible factor) by preventing proteasomal degradation. The transcription factor HIF increases the expression of angiogenesis regulated genes, such as VEGF, which can lead to high microvessel density and tumorigenesis. Yet this mechanism does not fully explain the large cytosolic population of fumarase molecules. We constructed a yeast strain in which fumarase is localized exclusively to mitochondria. This led to the discovery that the yeast cytosolic fumarase plays a key role in the protection of cells from DNA damage, particularly from DNA double-strand breaks. We show that the cytosolic fumarase is a member of the DNA damage response that is recruited from the cytosol to the nucleus upon DNA damage induction. This function of fumarase depends on its enzymatic activity, and its absence in cells can be complemented by high concentrations of fumaric acid. Our findings suggest that fumarase and fumaric acid are critical elements of the DNA damage response, which underlies the tumor suppressor role of fumarase in human cells and which is most probably HIF independent. This study shows an exciting crosstalk between primary metabolism and the DNA damage response, thereby providing a scenario for metabolic control of tumor propagation.

Topics: Cell Nucleus; Cytosol; DNA Damage; Fumarate Hydratase; Fumarates; Gene Knockdown Techniques; HeLa Cells; Histones; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Isoenzymes; Kidney Neoplasms; Leiomyomatosis; Mitochondria; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Tumor Suppressor Proteins

Individuals with hemizygous germline fumarate hydratase (FH) mutations are predisposed to renal cancer. These tumors predominantly exhibit functional inactivation of the remaining wild-type allele, implicating FH inactivation as a tumor-promoting event. Hypoxia-inducible factors are expressed in many cancers and are increased in clear cell renal carcinomas. Under normoxia, the HIFs are labile due to VHL-dependent proteasomal degradation, but stabilization occurs under hypoxia due to inactivation of HIF prolyl hydroxylase (HPH), which prevents HIF hydroxylation and VHL recognition. We demonstrate that FH inhibition, together with elevated intracellular fumarate, coincides with HIF upregulation. Further, we show that fumarate acts as a competitive inhibitor of HPH. These data delineate a novel fumarate-dependent pathway for regulating HPH activity and HIF protein levels.

Topics: Adult; Alleles; Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Renal Cell; DNA-Binding Proteins; Female; Fumarate Hydratase; Fumarates; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Ketoglutaric Acids; Kidney Neoplasms; Leiomyomatosis; Male; Middle Aged; Nuclear Proteins; Procollagen-Proline Dioxygenase; Syndrome; Transcription Factors; Up-Regulation