Fumarate Hydratase Inactivation Research Articles

Conditional Covalent Lethality Driven by Oncometabolite Accumulation.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a cancer predisposition syndrome driven by mutation of the tumor suppressor fumarate hydratase (FH). Inactivation of FH causes accumulation of the electrophilic oncometabolite fumarate. In the absence of methods for reactivation, tumor suppressors can be targeted via identification of synthetic lethal interactions using genetic screens. Inspired by recent advances in chemoproteomic target identification, here, we test the hypothesis that the electrophilicity of the HLRCC metabolome may produce unique susceptibilities to covalent small molecules, a phenomenon we term conditional covalent lethality. Screening a panel of chemically diverse electrophiles, we identified a covalent ligand, MP-1, that exhibits FH-dependent cytotoxicity. Synthesis and structure-activity profiling identified key molecular determinants underlying the molecule's effects. Chemoproteomic profiling of cysteine reactivity together with clickable probes validated the ability of MP-1 to engage an array of functional cysteines, including one lying in the Zn-finger domain of the tRNA methyltransferase enzyme TRMT1. TRMT1 overexpression rescues tRNA methylation from inhibition by MP-1 and partially attenuates the covalent ligand's cytotoxicity. Our studies highlight the potential for covalent metabolites and small molecules to synergistically produce novel synthetic lethal interactions and raise the possibility of applying phenotypic screening with chemoproteomic target identification to identify new functional oncometabolite targets.

Hereditary Leiomyomatosis and Renal Cell Cancer: Recent Insights Into Mechanisms and Systemic Treatment.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a rare autosomal dominant hereditary cancer syndrome characterized by a predisposition to cutaneous leiomyomas, uterine leiomyomas, and renal cell carcinoma (RCC). It is known to be caused by germline mutations of the fumarate hydratase (FH) gene, which encodes an enzyme component of the citric acid cycle and catalyzes the conversion of fumarate to L-malate. Currently, there is no standardized treatment for HLRCC, which may be due in part to a lack of understanding of the underlying mechanisms. Here, the underlying molecular mechanisms by which the inactivation of FH causes HLRCC are discussed. Additionally, potential therapeutic pharmacological strategies are also summarized to provide new perspectives for the prevention and treatment of HLRCC.

Computational Saturation Screen Reveals the Landscape of Mutations in Human Fumarate Hydratase.

Single amino acid substitutions within protein structures often manifest with clinical conditions in humans. The mutation of a single amino can significantly alter protein folding and stability, or change protein dynamics to influence function. The chemical engineering field has developed a large toolset for predicting the influence of point mutations with the aim of guiding the design of improved and more stable proteins. Here, we reverse this general protocol and adapt these tools for the prediction of damaging mutations within proteins. Mutations to fumarate hydratase (FH), an enzyme of the citric acid cycle, can lead to human diseases. The inactivation of FH by mutation causes leiomyomas and renal cell carcinoma by subsequent fumarate buildup and reduction in available malate. We present a scheme for accurately predicting the clinical effects of every possible mutation in FH by adaptation to a database of characterized damaging and benign mutations. Using energy prediction tools Rosetta and FoldX coupled with molecular dynamics simulations, we accurately predict individual mutations as well as mutational hotspots with a high disruptive capability in FH. Furthermore, through dynamic analysis, we find that hinge regions of the protein can be stabilized or destabilized by mutations, with mechanistic implications for the functional ability of the enzyme. Finally, we categorize all potential mutations in FH into functional groups, predicting which known mutations in the human population are loss of function, therefore having clinical implications, and validate our findings through metabolomics data of characterized human cell lines.

Systematic molecular and clinical analysis of uterine leiomyomas from fertile-aged women undergoing myomectomy.

What are the distributions and associated clinical characteristics of mediator complex subunit 12 (MED12), high mobility group AT-hook 2 (HMGA2) and fumarate hydratase (FH) aberrations in uterine leiomyomas from fertile-aged myomectomy patients? These driver mutations account for the majority (83%) of tumours in fertile-aged patients. Alterations affecting MED12, HMGA2 and FH account for 80-90% of uterine leiomyomas from middle-aged hysterectomy patients, while the molecular background of tumours from young myomectomy patients has not been systematically studied. A retrospective series of 361 archival uterine leiomyoma samples from 234 women aged ≤45 years undergoing myomectomy in 2009-2014 was examined. Associations between the molecular data and detailed clinical information of the patients and tumours were analysed. DNA was extracted from formalin-fixed paraffin-embedded samples and MED12 exons 1 and 2 were sequenced to identify mutations. Level of HMGA2 expression was evaluated by immunohistochemistry. Biallelic FH inactivation was analysed with 2-succinylcysteine staining, which is an indirect method of assessing FH deficiency. All patients' medical histories were reviewed, and clinical information of patients and tumours was combined with molecular data. The median age at operation was 34 years. The majority (58%) of patients were operated on for a single leiomyoma. Known driver mutations were identified in 83% of tumours (71% MED12; 9% HMGA2; 3% FH). In solitary leiomyomas, the MED12 mutation frequency was only 43%, and 29% were wild-type for all driver alterations. MED12 mutations were associated with multiple tumours, smaller tumour size and subserosal location. Although comprehensive, the study is retrospective in nature and all samples have been collected for routine diagnostic purposes. The use of paraffin-embedded samples and immunohistochemistry may have led to an underestimation of mutations. Due to the limited sample size and rarity of especially FH-deficient leiomyomas, the data are partly descriptive. The contribution of driver mutations in leiomyomas from young myomectomy patients is comparable to tumours obtained from hysterectomies of mostly middle-aged women. Our results support the earlier findings that MED12 mutations are associated with multiple tumours, smaller tumour size and subserosal location. The study emphasizes the distinct molecular background of solitary leiomyomas, and more research is needed to clarify the underlying causes of the notable proportion of wild-type leiomyomas. The study was supported by the Academy of Finland (307773), the Sigrid Jusélius Foundation, the Cancer Foundation Finland and the iCAN Digital Precision Cancer Medicine Flagship. The authors declare no conflicts of interest. N/A.

Results from a phase II study of bevacizumab and erlotinib in subjects with advanced hereditary leiomyomatosis and renal cell cancer (HLRCC) or sporadic papillary renal cell cancer.

5004 Background: HLRCC is a familial cancer syndrome associated with a type 2 papillary RCC (pRCC) variant. HLRCC is caused by germline mutations in the gene for the Krebs cycle enzyme fumarate hydratase (FH). FH inactivation results in VHL-independent upregulation of hypoxia inducible factor, a reliance on aerobic glycolysis, and activation of the NRF2 pathway, features also shared by some sporadic pRCC tumors. We hypothesized that the metabolic alterations underlying these tumors would be susceptible to targeted therapy with a combination of bevacizumab and erlotinib. Methods: Patients with advanced pRCC were eligible to enroll on this phase II study. To enrich for patients with FH deficiency, those with 1) HLRCC and 2) sporadic pRCC were enrolled into parallel, independent cohorts. All patients received bevacizumab 10 mg/kg IV every 2 weeks and erlotinib 150 mg orally daily. Patients who had received no more than two agents targeting the VEGFR pathway were included. Patients remained on treatment until unacceptable toxicity or progression. The primary endpoint was overall response rate (ORR); secondary endpoints were progression free survival (PFS) and duration of response. Results: A total of 83 patients with pRCC, including 42 in the HLRCC cohort and 41 in the sporadic cohort were enrolled on study. The majority of patients were IMDC intermediate risk (53/83, 64%) and 27 (33%) had at least one prior treatment. The ORR was 51% (42/83; 95% CI, 40 – 61) in all patients, 64% (27/42; 95% CI, 49 – 77) in the HLRCC cohort, and 37% (15/41; 95% CI, 24 – 52) in the sporadic cohort. The median PFS was 14.2 months (95% CI, 11.4 – 18.6) in all patients, 21.1 months (95% CI, 15.6 – 26.6) in the HLRCC cohort, and 8.7 months (95% CI, 6.4 – 12.6) in the sporadic cohort. The majority of treatment related adverse events (TRAEs) were grade 1 or 2 with the most common being acneiform rash (92%), diarrhea (77%), proteinuria (71%), and dry skin (61%). Grade ≥3 TRAEs occurred in 47% of patients, including hypertension (34%) and proteinuria (13%), with one patient (1.2%) with a grade 5 GI hemorrhage possibly related to bevacizumab. Conclusions: The combination of bevacizumab and erlotinib is well tolerated and is associated with encouraging activity in advanced pRCC, particularly in patients with FH deficient tumors. This is the first and largest prospective study in HLRCC and provides the basis for considering bevacizumab and erlotinib as a preferred option in a patient population that has no widely accepted standard. Clinical trial information: NCT01130519 .

Abstract SY19-01: Metabolic consequences and liabilities of TCA cycle truncation in cancer

Abstract Succinate dehydrogenase (SDH) and fumarate hydratase (FH) are tricarboxylic acid (TCA) cycle enzymes while SDH is also part of the respiratory chain. Despite their key housekeeping activities, the genetic loss of FH or of any of the SDH subunits is associated with susceptibility to cancers. SDH or FH inactivation leads to the accumulation of metabolites which cause the activation of hypoxia-inducible factors and the inhibition of histone and DNA demethylases, establishing pseudohypoxic and hypermethylator phenotype. However, surviving the loss of these essential metabolic enzymes requires a significant metabolic adaptation as backup mechanisms for the dysfunctional TCA cycle and oxidative phosphorylation in these tumors. Identifying these adaptations would reveal effective and specific metabolic vulnerabilities which can serve as potential molecular targets for therapeutic intervention. We have identified in the past a few metabolic essentialities in cells deficient in FH and SDH and now performed a pharmacologic phenotypic synthetic lethal screen to identified effective drugs that specifically kill SDH- and FH-deficient cells. Utilizing metabolomics, we have delineated the molecular target of the effective compounds. Citation Format: Eyal Gottlieb. Metabolic consequences and liabilities of TCA cycle truncation in cancer [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 SY19-01.

MED12 mutations and fumarate hydratase inactivation in uterine adenomyomas.

STUDY QUESTIONDo the uterine leiomyoma driver events – mediator complex subunit 12 (MED12) mutations, high mobility group AT-hook (HMGA2) overexpression, and fumarate hydratase (FH) inactivation – also contribute to the development of uterine adenomyomas?SUMMARY ANSWER MED12 mutations and FH deficiency occur in a subset of uterine adenomyomas, but at lower frequencies than in leiomyomas.WHAT IS KNOWN ALREADYUterine adenomyomas are benign tumours with clinical features very similar to uterine leiomyomas. Mutations affecting MED12, HMGA2 and FH account for up to 80–90% of leiomyomas, but their contribution to adenomyomas is not known.STUDY DESIGN, SIZE, DURATIONFormalin-fixed paraffin-embedded adenomyoma samples from 21 patients operated on during 2012–2014 were collected at the pathology department’s archives and analysed for uterine leiomyoma driver events.PARTICIPANTS/MATERIALS, SETTING, METHODSAdenomyoma diagnoses were verified by a specialized pathologist and representative areas were marked on haematoxylin-eosin slides. DNA was extracted from the tissue samples and sequenced to detect mutations in MED12. Expression levels of HMGA2 and 2SC, a robust indirect method to detect FH inactivation, were analysed by immunohistochemistry (IHC). The coding region of FH was sequenced in one adenomyoma sample showing strong 2SC staining as well as in the same patient’s normal tissue sample. All patients’ medical histories were collected and reviewed.MAIN RESULTS AND THE ROLE OF CHANCE MED12 mutation c.131G > A, p.G44D, the most common mutation in uterine leiomyomas, was identified in two samples (2/21; 9.5%). One adenomyoma displayed strong 2SC positivity and subsequent sequencing revealed a frameshift FH mutation c.911delC, p.P304fs in the tumour. The mutation was also present in the patient’s normal tissue sample, indicating that she has a hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome. HMGA2 protein expression was normal in all adenomyomas.LIMITATIONS, REASONS FOR CAUTIONRestricted sample size limits the determination of exact mutation frequencies of the studied aberrations in adenomyomas.WIDER IMPLICATIONS OF THE FINDINGSUterine leiomyoma driver mutations do contribute to the development of some adenomyomas. We also report an adenomyoma in the context of hereditary HLRCC syndrome. Despite clinical similarities, the pathogenic mechanisms of adenomyomas and leiomyomas are likely different. Large-scale genomic analyses are warranted to elucidate the complete molecular background of adenomyomas.STUDY FUNDING/COMPETING INTERESTSThis study was supported by The Academy of Finland, the Sigrid Jusélius Foundation, and the Cancer Society of Finland. The authors declare no conflict of interest.

Fumarate Hydratase Mutations and Alterations in Leiomyoma With Bizarre Nuclei

Leiomyoma with bizarre nuclei (LM-BN), is a variant of uterine smooth muscle tumor with atypical histologic features. Although some LM-BN share several significant genetic alterations with leiomyosarcoma, including p16 and p53, the underlying tumorigenesis of LM-BN remains largely unknown. As we previously reported, LM-BN can be divided into 2 subtypes, type I and type II, based on different nuclear features. Type I LM-BN have similar histologic features as uterine smooth muscle tumors with fumarate hydratase (FH) alterations. In this study, we examined FH expression and FH mutations in 77 LM-BN (40 type I cases and 37 type II cases). FH expression was examined by immunohistochemistry using S-(2-succino)-cysteine antibodies (2SC, a protein modification associated with FH inactivation and subsequent fumarate accumulation) and FH antibodies (FH gene products). Seventy-two LM-BN tumors underwent Sanger sequencing to detect FH mutations. We found that 51% (39/77) of LM-BN showed FH alterations detected by immunohistochemistry with both 2SC and FH. Mutational analysis showed that 21% (15/72) of LM-BN harbored FH gene mutations. Further analysis revealed that 85% (34/40) of those with FH alterations were type I LM-BN while 19% (7/37) were type II LM-BN. Our findings suggest that over half of histologically diagnosed LM-BN may be related to FH alterations or FH mutations and the majority of these have the characteristic histologic features of type I LM-BN.

Fumarate hydratase inactivation in hereditary leiomyomatosis and renal cell cancer is synthetic lethal with ferroptosis induction.

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a hereditary cancer syndrome characterized by inactivation of the Krebs cycle enzyme fumarate hydratase (FH). HLRCC patients are at high risk of developing kidney cancer of type 2 papillary morphology that is refractory to current radiotherapy, immunotherapy and chemotherapy. Hence, an effective therapy for this deadly form of cancer is urgently needed. Here, we show that FH inactivation (FH −/−) proves synthetic lethal with inducers of ferroptosis, an iron‐dependent and nonapoptotic form of cell death. Specifically, we identified gene signatures for compound sensitivities based on drug responses for 9 different drug classes against the NCI‐60 cell lines. These signatures predicted that ferroptosis inducers would be selectively toxic to FH −/− cell line UOK262. Preferential cell death against UOK262‐FH −/− was confirmed with 4 different ferroptosis inducers. Mechanistically, the FH −/− sensitivity to ferroptosis is attributed to dysfunctional GPX4, the primary cellular defender against ferroptosis. We identified that C93 of GPX4 is readily post‐translationally modified by fumarates that accumulate in conditions of FH −/−, and that C93 modification represses GPX4 activity. Induction of ferroptosis in FH‐inactivated tumors represents an opportunity for synthetic lethality in cancer.

Abstract 4611: Uterine leiomyoma driver events in uterine adenomyomas

Abstract Uterine adenomyosis is a condition in which ectopic endometrial glands are present in myometrial stroma surrounded by smooth muscle cell hyperplasia. Foci of adenomyosis growing as a tumor like mass are called adenomyomas. Uterine adenomyomas are common tumors and they share symptoms, including pelvic pain and abnormal bleeding, with uterine leiomyomas. The two tumor types are challenging to distinguish from one another and the diagnosis is usually confirmed only after surgery by pathological evaluation. The molecular background of uterine adenomyomas is not currently well known. In uterine leiomyomas, somatic mediator complex subunit 12 (MED12) mutations, high mobility group AT-hook (HMGA2) protein overexpression, and fumarate hydratase (FH) inactivation are well established as major mutually exclusive driver events covering 80-90% of the tumors. Here, we have analyzed the presence of these changes in a set of 21 uterine adenomyomas. Representative areas of formalin-fixed paraffin embedded archival uterine adenomyoma tissue samples were used to construct a tissue microarray. The HMGA2 overexpression and the FH inactivation were assessed using immunohistochemistry with anti HMGA and 2SC antibodies, respectively. DNA was extracted from the tumor samples to determine the MED12 mutation status by direct sequencing of exons 1 and 2 of the gene. MED12 c.131GA, p.G44D mutation was found in two adenomyoma samples out of 21 (9.5%). Strong positive staining of 2SC indicating FH inactivation was present in one sample which also showed reduced FH protein expression when validated with an independent method using anti-FH immunostaining. Sequencing revealed a frameshift mutation c.911delC, p.P304fs in exon 7 leading to a premature stop codon 25 codons later. The mutation was also found in a separate uterine leiomyoma of the same patient and both tumor samples mostly presented the mutant allele indicating loss of heterozygosity of the wild type allele. This, together with the patient's medical history of previous uterine leiomyomas, indicates the germline origin of the mutation and thus a hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome. No changes in HMGA2 expression were detected with all samples presenting normal expression levels. In conclusion, MED12 mutations are present in a subset of uterine adenomyomas. The mutation frequency of 9.5% that was observed here in our adenomyoma sample series is considerably lower than that of 70% in uterine leiomyomas. Our results also suggest that adenomyomas may be linked to HLRCC in which they have not been previously reported. The driver events behind uterine adenomyomas remain mostly unknown and further large-scale studies are warranted to clarify the spectrum of underlying mutations and molecular background of these common tumors. Citation Format: Tuomas A. Heikkinen, Anna Äyräväinen, Janne Hänninen, Terhi Ahvenainen, Ralf Bützow, Annukka Pasanen, Pia Vahteristo. Uterine leiomyoma driver events in uterine adenomyomas [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 4611.

Abstract 1457: Somatic biallelic inactivation of fumarate hydratase (FH) in uterine leiomyomas

Abstract Uterine leiomyomas (ULMs) are highly common benign tumors that affect the health of millions of women by causing a variety of symptoms. Furthermore, ULMs are the most common indication for hysterectomy, generating considerable economic costs for the health care system. Recurrent and mutually exclusive genetic aberrations leading to unique gene expression profiles have been identified in ULMs. Thus these tumors can be divided into distinct subclasses according to their molecular genetic background. The majority of ULMs (80-90%) have specific mutations in MED12 or aberrations in HMGA2. Somatic biallelic inactivation of FH can also, albeit rarely, drive ULM tumorigenesis and has previously been detected in 1.3% of sporadic ULM samples (2/153; Lehtonen et al, 2004). FH encodes the fumarase enzyme that catalyzes the hydration of fumarate to L-malate in the tricarboxylic acid cycle. Biallelic inactivation of FH leads to accumulation of fumarate and aberrant succination of proteins. Heterozygous germline FH mutations predispose to a rare autosomal dominant tumor predisposition syndrome, hereditary leiomyomatosis and renal cell cancer (HLRCC). This highly penetrant syndrome is characterized by uterine and cutaneous leiomyomas and in some cases renal cell cancer. In this study, we re-evaluated the frequency of somatic biallelic inactivation of FH in ULMs in the largest sample set thus far. The study material consisted of 1167 fresh frozen ULM and their respective normal myometrium tissue samples from 375 hysterectomy patients. All samples were analyzed with high-throughput SNP array (Infinium Human Core-24+ Kit, Illumina Inc., San Diego, CA). All tumors detected with somatic deletions or loss of heterozygosity (LOH) in the FH locus were included in S-(2-succinyl) cysteine (2SC) immunohistochemistry (IHC) to indirectly detect biallelic FH inactivation. Also samples without FH double deletions were selected for Sanger sequencing of FH coding regions to detect inactivating mutations. Somatic FH deletions or LOH were detected in 30 tumors, of which 10 were identified FH deficient by 2SC IHC. In these samples, another deletion (a double deletion) in three ULMs and a somatic nucleotide change in six ULMs was found to be the second hit causing biallelic inactivation of FH. In one sample no second hit was detected with these methods. From the six somatic nucleotide changes, four were different FH missense mutations predicted to be damaging (SIFT, PolyPhen-2, Provean) and two were synonymous substitutions in the last nucleotide of different exons. Studies to determine the impact of these synonymous changes on splicing are underway. To conclude, in our sample set we detected 0.9% (10/1167) of the ULMs to harbour somatic biallelic inactivation of FH. MED12 exon 1 or 2 mutations and HMGA2 rearrangements were not detected in these tumors. Thus the previously published results were validated in this study with the largest sample set so far. Citation Format: Jaana Tolvanen, Netta Mäkinen, Hanna-Riikka Heinonen, Simona Bramante, Miika Mehine, Nanna Sarvilinna, Jari Sjöberg, Oskari Heikinheimo, Annukka Pasanen, Ralf Bützow, Lauri A. Aaltonen. Somatic biallelic inactivation of fumarate hydratase (FH) in uterine leiomyomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1457. doi:10.1158/1538-7445.AM2017-1457

Abstract 2461: Molecular classification and clinical characterization of a large uterine leiomyoma patient cohort

Abstract Uterine leiomyomas (ULs), benign smooth muscle tumors, represent one of the most common neoplasms in women with an estimated prevalence varying from 20% to over 70% during the reproductive years. Approximately every fourth woman with ULs has clinically relevant lesions, which cause morbidity and thus require treatment. Still today, hysterectomy is the primary treatment option for ULs worldwide, and remarkably, their annual societal costs exceed those of colon and breast cancer combined. Our previous findings, derived from the use of high-throughput technologies, suggest that there are at least three distinct molecular UL subclasses, each displaying a characteristic genetic driver aberration and unique global gene expression profile: MED12 (mediator complex subunit 12) mutation-positive, HMGA2 (high mobility group AT-hook 2)-overexpressing, and FH (fumarate hydratase)-deficient ULs. The aim of this study is to examine the molecular subclasses, their respective proportions, and clinical characteristics in a large UL patient cohort. The study material consists of 1026 ULs and corresponding normal myometrium tissue from 322 patients, who had ultrasound-diagnosed ULs and underwent hysterectomy in Helsinki University Hospital, Finland between October 2013 and June 2016. From each uterus, we harvested all feasible ULs ≥1 cm in diameter and a piece of the corresponding normal myometrium tissue. The location of the collected samples in the uterus, their size, and any observed special characteristics were carefully documented at the time of sample removal. In addition, comprehensive clinical information of the patients was obtained from medical and pathology reports, as well as from a questionnaire, and the histopathology of all lesions were characterized according to the WHO 2014 criteria. All collected ULs have been systematically screened for MED12 mutations by Sanger sequencing. Overexpression of HMGA2 typically arises from a chromosomal translocation, and deletions at FH locus suggest the presence of potential biallelic FH inactivation. Therefore, HMGA2-overexpressing and FH-deficient ULs have tentatively been identified using a high-density customizable Infinium® HumanCore-24+ BeadChip with over 305,000 markers. Currently, the data analyses are ongoing. Sufficient numbers of well-documented high-quality samples are a prerequisite for successful research. We hypothesize that ULs can emerge through several distinct molecular mechanisms that contribute to different biological and clinicopathological features and response to treatment. Their molecular classification and further characterization will lead to increased knowledge on UL genesis as well as improved management of the disease. Citation Format: Netta Mäkinen, Hanna-Riikka Heinonen, Annukka Pasanen, Jaana Tolvanen, Simona Bramante, Miika Mehine, Jari Sjöberg, Oskari Heikinheimo, Ralf Bützow, Lauri A. Aaltonen. Molecular classification and clinical characterization of a large uterine leiomyoma patient cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2461. doi:10.1158/1538-7445.AM2017-2461

MED12 mutations and FH inactivation are mutually exclusive in uterine leiomyomas.

Background:Uterine leiomyomas from hereditary leiomyomatosis and renal cell cancer (HLRCC) patients are driven by fumarate hydratase (FH) inactivation or occasionally by mediator complex subunit 12 (MED12) mutations. The aim of this study was to analyse whether MED12 mutations and FH inactivation are mutually exclusive and to determine the contribution of MED12 mutations on HLRCC patients' myomagenesis.Methods:MED12 exons 1 and 2 mutation screening and 2SC immunohistochemistry indicative for FH deficiency was performed on a comprehensive series of HLRCC patients' (122 specimens) and sporadic (66 specimens) tumours. Gene expression analysis was performed using Affymetrix GeneChip Human Exon Arrays (Affymetrix, Santa Clara, CA, USA).Results:Nine tumours from HLRCC patients harboured a somatic MED12 mutation and were negative for 2SC immunohistochemistry. All remaining successfully analysed lesions (107/116) were deficient for FH. Of sporadic tumours, 35/64 were MED12 mutation positive and none displayed a FH defect. In global gene expression analysis FH-deficient tumours clustered together, whereas HLRCC patients' MED12 mutation-positive tumours clustered together with sporadic MED12 mutation-positive tumours.Conclusions:Somatic MED12 mutations and biallelic FH inactivation are mutually exclusive in both HLRCC syndrome-associated and sporadic uterine leiomyomas. The great majority of HLRCC patients' uterine leiomyomas are caused by FH inactivation, but incidental tumours driven by somatic MED12 mutations also occur. These MED12 mutation-positive tumours display similar expressional profiles with their sporadic counterparts and are clearly separate from FH-deficient tumours.

Inactivation of SDH and FH cause loss of 5hmC and increased H3K9me3 in paraganglioma/pheochromocytoma and smooth muscle tumors.

Succinate dehydrogenase (SDH) and fumarate hydratase (FH) are tricarboxylic acid (TCA) cycle enzymes and tumor suppressors. Loss-of-function mutations give rise to hereditary paragangliomas/pheochromocytomas and hereditary leiomyomatosis and renal cell carcinoma. Inactivation of SDH and FH results in an abnormal accumulation of their substrates succinate and fumarate, leading to inhibition of numerous α-ketoglutarate dependent dioxygenases, including histone demethylases and the ten-eleven-translocation (TET) family of 5-methylcytosine (5 mC) hydroxylases. To evaluate the distribution of DNA and histone methylation, we used immunohistochemistry to analyze the expression of 5 mC, 5-hydroxymethylcytosine (5 hmC), TET1, H3K4me3, H3K9me3, and H3K27me3 on tissue microarrays containing paragangliomas/pheochromocytomas (n = 134) and hereditary and sporadic smooth muscle tumors (n = 56) in comparison to their normal counterparts. Our results demonstrate distinct loss of 5 hmC in tumor cells in SDH- and FH-deficient tumors. Loss of 5 hmC in SDH-deficient tumors was associated with nuclear exclusion of TET1, a known regulator of 5 hmC levels. Moreover, increased methylation of H3K9me3 occurred predominantly in the chief cell component of SDH mutant tumors, while no changes were seen in H3K4me3 and H3K27me3, data supported by in vitro knockdown of SDH genes. We also show for the first time that FH-deficient smooth muscle tumors exhibit increased H3K9me3 methylation compared to wildtype tumors. Our findings reveal broadly similar patterns of epigenetic deregulation in both FH- and SDH-deficient tumors, suggesting that defects in genes of the TCA cycle result in common mechanisms of inhibition of histone and DNA demethylases.

Fumarate induces redox-dependent senescence by modifying glutathione metabolism.

Mutations in the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) are associated with a highly malignant form of renal cancer. We combined analytical chemistry and metabolic computational modelling to investigate the metabolic implications of FH loss in immortalized and primary mouse kidney cells. Here, we show that the accumulation of fumarate caused by the inactivation of FH leads to oxidative stress that is mediated by the formation of succinicGSH, a covalent adduct between fumarate and glutathione. Chronic succination of GSH, caused by the loss of FH, or by exogenous fumarate, leads to persistent oxidative stress and cellular senescence in vitro and in vivo. Importantly, the ablation of p21, a key mediator of senescence, in Fh1-deficient mice resulted in the transformation of benign renal cysts into a hyperplastic lesion, suggesting that fumarate-induced senescence needs to be bypassed for the initiation of renal cancers.

Abstract 3514: MED12 and FH mutations in HLRCC associated uterine leiomyomas

Abstract Uterine leiomyomas (fibroids) are benign tumors which originate from the smooth muscle cells of the myometrium. Almost 70% of women are affected by these tumors by the age of 50. Even though uterine leiomyomas are benign, they can cause difficult symptoms and are the most common indication for hysterectomy. Most uterine leiomyomas are sporadic and up to 70% of these lesions harbor very specific mutations in MED12 (mediator complex subunit 12). All MED12 mutations observed in uterine leiomyomas have been missense or insertion deletion mutations retaining the open reading frame. Uterine leiomyomas occur also in the context of Hereditary Leiomyomatosis and Renal Cell Cancer syndrome (HLRCC, OMIM # 150800). In addition to uterine leiomyomas, the syndrome predisposes individuals to cutaneous leiomyomas and renal cell cancer. HLRCC is an autosomal dominant syndrome caused by heterozygous germline mutations in FH (fumarate hydratase). The gene is classified as a tumor suppressor and according to Knudson's two-hit hypothesis, syndrome related tumors display biallelic inactivation of FH. Recently, we reported 3/34 uterine leiomyomas from HLRCC patients to harbor somatic mutations in MED12. Loss of heterozygosity (LOH) at the FH locus was assessed from the tumors, and interestingly none of the MED12 mutation positive tumors showed biallelic inactivation of FH through LOH. Exome sequencing of seemingly sporadic uterine leiomyomas revealed also one patient to carry a truncating germline mutation in FH. The FH and MED12 loci from the uterine leiomyomas of this patient were further studied with direct sequencing and LOH assessment. Tumors had different MED12 mutations and none of them harbored a somatic mutation or displayed LOH as a second hit at FH locus. The aim of this study is to clarify the role of MED12 in HLRCC syndrome associated uterine leiomyomas and to analyze whether MED12 mutations and biallelic inactivation of FH are truly mutually exclusive. A tissue microarray including uterine leiomyomas from HLRCC patients will be constructed and FH inactivation will be analyzed with immunohistochemical stainings. Expression analyses of the tumors harboring somatic MED12 mutation and FH mutation in germline will show whether these tumors cluster together with MED12 mutation positive tumors, tumors with biallelic FH inactivation, or if they form a separate cluster. Results of these analyses will reveal if there are two distinct molecular mechanisms behind the development of uterine leiomyomas in HLRCC patients; one dependent on the biallelic inactivation of FH and the other on the altered function of MED12. Citation Format: Kati Kämpjärvi, Netta Mäkinen, Miika Mehine, Jaana Tolvanen, Tuomas Heikkinen, Ralf Bützow, Lauri A. Aaltonen, Pia Vahteristo. MED12 and FH mutations in HLRCC associated uterine leiomyomas. [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 3514. doi:10.1158/1538-7445.AM2014-3514

Metabolic Reprogramming for Producing Energy and Reducing Power in Fumarate Hydratase Null Cells from Hereditary Leiomyomatosis Renal Cell Carcinoma

Fumarate hydratase (FH)-deficient kidney cancer undergoes metabolic remodeling, with changes in mitochondrial respiration, glucose, and glutamine metabolism. These changes represent multiple biochemical adaptations in glucose and fatty acid metabolism that supports malignant proliferation. However, the metabolic linkages between altered mitochondrial function, nucleotide biosynthesis and NADPH production required for proliferation and survival have not been elucidated. To characterize the alterations in glycolysis, the Krebs cycle and the pentose phosphate pathways (PPP) that either generate NADPH (oxidative) or do not (non-oxidative), we utilized [U-13C]-glucose, [U-13C,15N]-glutamine, and [1,2- 13C2]-glucose tracers with mass spectrometry and NMR detection to track these pathways, and measured the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of growing cell lines. This metabolic reprogramming in the FH null cells was compared to cells in which FH has been restored. The FH null cells showed a substantial metabolic reorganization of their intracellular metabolic fluxes to fulfill their high ATP demand, as observed by a high rate of glucose uptake, increased glucose turnover via glycolysis, high production of glucose-derived lactate, and low entry of glucose carbon into the Krebs cycle. Despite the truncation of the Krebs cycle associated with inactivation of fumarate hydratase, there was a small but persistent level of mitochondrial respiration, which was coupled to ATP production from oxidation of glutamine-derived α–ketoglutarate through to fumarate. [1,2- 13C2]-glucose tracer experiments demonstrated that the oxidative branch of PPP initiated by glucose-6-phosphate dehydrogenase activity is preferentially utilized for ribose production (56-66%) that produces increased amounts of ribose necessary for growth and NADPH. Increased NADPH is required to drive reductive carboxylation of α-ketoglutarate and fatty acid synthesis for rapid proliferation and is essential for defense against increased oxidative stress. This increased NADPH producing PPP activity was shown to be a strong consistent feature in both fumarate hydratase deficient tumors and cell line models.

Abstract 1998: Exome sequencing and SNP array analysis of HLRCC syndromic uterine leiomyomas.

Abstract Uterine leiomyomas (fibroids) affect approximately 70% of women by the age of 50 years and thus are the most frequent tumors of the female reproductive tract. Fibroids are benign tumors originating from the smooth muscle cells of the uterus. They may cause various symptoms including abdominal pain, abnormal bleeding, and pregnancy complication, and they are the most common cause for hysterectomy. Our recent study identified somatic mutations in mediator complex subunit 12 (MED12) in as many as 70% of sporadic fibroids. Traditionally, fibroids have been considered rather stable, although some recurrent cytogenetic rearrangements affecting mainly chromosomes 7q, 12q15, and 6p21 have been reported. Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC, OMIM 150800) is an autosomal dominant cancer syndrome which predisposes to cutaneous and uterine leiomyomas, and renal cell cancer. Syndrome is caused by heterozygous germline mutations in the tumor suppressor gene fumarate hydratase (FH) at 1q42.1. The gene encodes fumarase, which catalyzes the hydratation of fumarate to L-malate in mitochondrial tricarboxylic acid cycle. Cutaneous and uterine leiomyomas are the most prevalent lesions with the syndrome and present typically at the age of 25 to 30 years. The aim of this study was to systematically characterize the genomic profile of FH deficient fibroids. For this, we analyzed altogether 10 fibroids and their corresponding myometrial samples from two Finnish HLRCC-family members and one sporadic leiomyoma sample with two somatic changes in FH by exome sequencing and SNP array analyses. To identify somatic variants, exome data from the tumors was filtered against the data from the respective myometrial samples as well as 93 Finnish individuals from the 1000 Genomes Project, 62 in-house controls, and all known variants in the dbSNP database. Altogether only 18 validated somatic mutations were observed in the exome data. All the variants were unique; they were observed only in one sample and the respective genes did not harbor additional changes in other tumors. No variations in genes commonly mutated in malignant tumors e.g. TP53, Rb or PTEN were observed. Neither were MED12 mutations, suggesting that mutations in MED12 and biallelic FH inactivation are mutually exclusive. In the SNP array analysis most of the tumors were found to carry large (>5 Mb) aberrations. The only recurrent change was the deletions at the FH locus. Deletions at 7q, which are frequently observed in sporadic uterine leiomyomas, were not present in these FH deficient tumors. To summarize, exome sequencing and SNP array analyses of 11 FH deficient tumors did not reveal any recurrent somatic variations in addition to the changes at the site of FH at 1q42. Loss of FH function damages the tricarboxylic acid cycle and causes a severe metabolic stress for the cells. This may be sufficient to induce formation of these benign lesions of the uterus. Citation Format: Kati Kämpjärvi, Miika Mehine, Pia Vahteristo, Lauri A. Aaltonen. Exome sequencing and SNP array analysis of HLRCC syndromic uterine leiomyomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1998. doi:10.1158/1538-7445.AM2013-1998

MED12 exon 2 mutations in histopathological uterine leiomyoma variants

Uterine leiomyomas, or fibroids, are the most common human tumors. Based on histopathology, they can be divided into common leiomyomas and various relatively rare subtypes that mimic malignancy in one or more aspects. Recently, we showed that exon 2 of mediator complex subunit 12 (MED12) is mutated in up to 70% of common fibroids. To investigate the frequency of MED12 exon 2 mutations in histopathological uterine leiomyoma variants, we screened altogether 206 lesions, including 69 histopathologically common leiomyomas, 59 cellular (23 cellular and 36 highly cellular), 18 atypical and 26 mitotically active leiomyomas, as well as 34 uterine fibroid samples from 14 hereditary leiomyomatosis and renal cell cancer patients with a heterozygous germ line mutation in fumarate hydratase (FH). The uterine leiomyoma variants harbored MED12 exon 2 mutations significantly less frequently than common leiomyomas (P=2.93 × 10(-8)). In all, 6 mutations were detected among cellular fibroids (6/67; 8.96%), 3 among atypical fibroids (3/18; 16.67%) and 10 among mitotically active fibroids (10/26; 38.46%). Only mitotically active fibroids displayed a mutation frequency that was not statistically different from common leiomyomas (P=0.11). Three MED12 exon 2 mutations were detected among 34 tumors with a heterozygous germ line FH mutation (P=5.28 × 10(-7)). None of these tumors displayed biallelic inactivation of FH. Our results suggest that MED12 mutation positivity is a key characteristic of common leiomyomas. Cellular and atypical fibroids, in particular, may arise through different molecular mechanisms. The results also propose that MED12 and biallelic FH mutations may be mutually exclusive.

Fumarate hydratase inactivation in renal tumors: HIF1 alpha, NRF2, and "cryptic targets" of transcription factors

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).