Von Hippel-Lindau Gene Research Articles

Central Nervous System Hemangioblastoma in a Pediatric Patient Associated With Von Hippel-Lindau Disease: A Case Report and Literature Review.

BackgroundHemangioblastoma is a benign tumor of the central nervous system and may appear as a component of von Hippel-Lindau (VHL) disease. At present, approximately 40 cases of optic nerve HGBs have been reported in the literature. VHL disease is a rare autosomal-dominant inherited cancer syndrome with different phenotypes caused by variants in the VHL gene. Herein, the authors describe a case of a pediatric patient with VHL disease and with optic nerve HGB, a rare phenotypic expression. The purpose of this study was to explore the genotype-phenotype, clinical features, treatment and follow-up of VHL-associated hemangioblastomas in pediatric patients.Case DescriptionA 12-year-old boy presented with vision loss, headache and dizziness at our hospital. Magnetic resonance imaging (MRI) revealed a large (19.8 mm*18.5 mm*23.5 mm) irregular mass located in the suprasellar region. The mass was successfully removed after craniotomy and microsurgical treatment. The pathological diagnosis was left optic nerve HGB. Genetic analyses showed p.Pro86Leu (c. 257C>T) heterozygous missense mutations in the VHL gene.ConclusionThis is the first reported pediatric case of VHL-associated optic nerve HGB. The genotype-phenotype correlation of VHL disease may provide new evidences for predicting tumor penetrance and survival. Gross tumor resection combined with stereotactic radiosurgery might be the most beneficial treatment.

Constellation of Von Hippel-Lindau Disease-Related Findings on a 68Ga-DOTATATE PET/CT.

Von Hippel-Lindau (VHL) is a rare predominantly hereditary syndrome characterized by multiple benign and malignant tumors that can affect different organ systems. We present representative images of a 68Ga-DOTATATE PET/CT in a patient with confirmed VHL gene mutation, which demonstrates a constellation of findings commonly seen in these patients in one single imaging modality.

Oxygen-Mediated Suppression of CD8+ T Cell Proliferation by Macrophages: Role of Pharmacological Inhibitors of HIF Degradation.

Myeloid cell interactions with cells of the adaptive immune system are an essential aspect of immunity. A key aspect of that interrelationship is its modulation by the microenvironment. Oxygen is known to influence myelosuppression of T cell activation in part via the Hypoxia inducible (HIF) transcription factors. A number of drugs that act on the HIF pathway are currently in clinical use and it is important to evaluate how they act on immune cell function as part of a better understanding of how they will influence patient outcomes. We show here that increased activation of the HIF pathway, either through deletion of the negative regulator of HIF, the von Hippel-Lindau (VHL) gene, in myeloid cells, or through pharmacological inhibitors of VHL-mediated degradation of HIF, potently suppresses T cell proliferation in myeloid cell/T cell culture. These data demonstrate that both pharmacological and genetic activation of HIF in myeloid cells can suppress adaptive cell immune response.

MK-6482 as a potential treatment for von Hippel-Lindau disease-associated clear cell renal cell carcinoma

ABSTRACT Introduction Von Hippel-Lindau (VHL) disease is an inherited autosomal dominant syndrome caused by a germline mutation and/or deletion of the VHL gene. Inappropriate hypoxia-inducible factor (HIF)-mediated transcription of proangiogenic and metabolic genes leads to the development of tumors and cysts in multiple organs. Surgery is a standard treatment for localized tumors with a risk of metastasis or organ dysfunction. Repeated surgeries cause substantial morbidity and have a major impact on quality of life. There is an urgent need to develop effective and safe systemic treatments for VHL disease manifestations. The small-molecule HIF 2 alpha inhibitor MK-6482 (belzutifan) has demonstrated significant efficacy in VHL disease related renal cell carcinomas, hemangioblastomas, and pancreatic neuroendocrine tumors while demonstrating an acceptable safety profile. Areas covered This paper reviews the development of the HIF-2 alpha inhibitor, MK-6482, and discusses preliminary results of ongoing phase I/II studies in renal cell carcinoma (RCC) and VHL disease. An examination of ongoing clinical development of MK-6482 and perspectives on potential future developments and challenges are offered. Expert opinion Because of its favorable safety profile, its clear efficacy in VHL disease, promising findings in sporadic, advanced RCC, and convenient oral formulation, MK-6482 is expected to become a leading treatment for VHL disease. Among other currently available oral agents, we believe that MK-6482 will be a preferred treatment for VHL-associated RCC.

Whole-Exome Sequencing Reveals Novel Variations in Patients with Familial Von Hippel−Lindau Syndrome

Von Hippel-Lindau (VHL) syndrome is a rare disease that occurs in an autosomal-dominant genetic pattern. Due to the high genetic variability of VHL diseases, current studies have limited clinical value. Moreover, casual genetic variations in patients with VHL syndrome are still unclear. Here, we performed whole-exome sequencing of 25 individuals to identify reliable disease-related variations. Systemic computational analysis was performed for variant detection, and Sanger sequencing was used to validate detected mutations. Most of the known mutations in the VHL gene were observed in the studied population. In addition, a large fragment deletion in VHL exon 2 in the immediate family members of the last family was detected. This had not been reported earlier. Moreover, we identified 3 novel mutation sites in the MAP2K3 gene that may be involved in the occurrence and development of the VHL disease. These results demonstrated that the heterogeneous nature of VHL syndrome and novel mutational signatures may help to improve the diagnostic ability of VHL syndrome.

Comprehensive characterization of Alu-mediated breakpoints in germline VHL gene deletions and rearrangements in patients from 71 VHL families.

Von Hippel‐Lindau (VHL) is a hereditary multisystem disorder caused by germline alterations in the VHL gene. VHL patients are at risk for benign as well as malignant lesions in multiple organs including kidney, adrenal, pancreas, the central nervous system, retina, endolymphatic sac of the ear, epididymis, and broad ligament. An estimated 30%–35% of all families with VHL inherit a germline deletion of one, two, or all three exons. In this study, we have extensively characterized germline deletions identified in patients from 71 VHL families managed at the National Cancer Institute, including 59 partial (PD) and 12 complete VHL deletions (CD). Deletions that ranged in size from 1.09 to 355 kb. Fifty‐eight deletions (55 PD and 3 CD) have been mapped to the exact breakpoints. Ninety‐five percent (55 of 58) of mapped deletions involve Alu repeats at both breakpoints. Several novel classes of deletions were identified in this cohort, including two cases that have complex rearrangements involving both deletion and inversion, two cases with inserted extra Alu‐like sequences, six cases that involve breakpoints in Alu repeats situated in opposite orientations, and a “hotspot” PD of Exon 3 observed in 12 families that involves the same pair of Alu repeats.

Molecular and Metabolic Subtypes in Sporadic and Inherited Clear Cell Renal Cell Carcinoma.

The promise of personalized medicine is a therapeutic advance where tumor signatures obtained from different omics platforms, such as genomics, transcriptomics, proteomics, and metabolomics, in addition to environmental factors including metals and metalloids, are used to guide the treatments. Clear cell renal carcinoma (ccRCC), the most common type of kidney cancer, can be sporadic (frequently) or genetic (rare), both characterized by loss of the von Hippel-Lindau (VHL) gene that controls hypoxia inducible factors. Recently, several genomic subtypes were identified with different prognoses. Transcriptomics, proteomics, metabolomics and metallomic data converge on altered metabolism as the principal feature of the disease. However, in view of multiple biochemical alterations and high level of tumor heterogeneity, identification of clearly defined subtypes is necessary for further improvement of treatments. In the future, single-cell combined multi-omics approaches will be the next generation of analyses gaining deeper insights into ccRCC progression and allowing for design of specific signatures, with better prognostic/predictive clinical applications.

Multiple Hepatic and Pulmonary Tumors Combined with Bilateral Adrenal Masses in a Young Woman with von Hippel–Lindau Disease

Question: A 37-year-old woman was admitted to our center for adrenal diseases with bilateral adrenal tumors and multiple hepatic and pulmonary lesions for surgery. She was first diagnosed with von Hippel–Lindau (VHL) syndrome in April 2000, with proof of heterozygous point mutation c.292T>C in exon 1 of the VHL gene. Her father was diagnosed with a VHL gene mutation as well. She complained of a 4- to 5-kg weight loss in 4 months, increased daytime fatigue, and anxiety. She reported occasional nicotine abuse in the past.

Ginsenoside 20(S)-Rg3 upregulates tumor suppressor VHL gene expression by suppressing DNMT3A-mediated promoter methylation in ovarian cancer cells

To explore the mechanism by which ginsenoside 20(S)-Rg3 upregulates the expression of tumor suppressor von Hippel-Lindau (VHL) gene in ovarian cancer cells. Ovarian cancer cell line SKOV3 treated with 20(S)-Rg3 were examined for mRNA and protein levels of VHL, DNMT1, DNMT3A and DNMT3B by real-time PCR and Western blotting, respectively. The changes in VHL mRNA expression in SKOV3 cells in response to treatment with 5-Aza-CdR, a DNA methyltransferase inhibitor, were detected using real-time PCR. VHL gene promoter methylation was examined with methylation-specific PCR and VHL expression levels were determined with real-time PCR and Western blotting in non-treated or 20(S)-Rg3-treated SKOV3 cells and in 20(S)-Rg3-treated DNMT3A-overexpressing SKOV3 cells. VHL and DNMT3A protein levels were detected by immunohistochemistry in subcutaneous SKOV3 cell xenografts in nude mice. Treatment of SKOV3 cells with 20(S)-Rg3 significantly upregulated VHL and downregulated DNMT3A expressions at both the mRNA and protein levels (P < 0.05) and upregulated DNMT3B expression only at the mRNA level, but did not cause significant changes in either the mRNA or protein level of DNMT1. Treatment of the cells with 2 and 5 μmol/L 5-Aza-CdR obviously increased VHL mRNA expression by by over 3 folds (P < 0.05). 20(S)-Rg3 significantly decreased the methylation level in the promoter region of VHL gene, and this effect was abrogated by DNMT3A overexpression in the cells (P < 0.05). Immunohistochemisty showed a significantly increased VHL expression but a lowered DNMT3A expression in subcutaneous SKOV3 cell xenografts in 20 (S)-Rg3-treated nude mice. Ginsenoside 20(S)-Rg3 upregulates VHL expression in ovarian cancer cells by suppressing DNMT3A-mediated DNA methylation.

Therapeutic inhibition of HIF-2α reverses polycythemia and pulmonary hypertension in murine models of human diseases

Polycythemia and pulmonary hypertension are 2 human diseases for which better therapies are needed. Upregulation of hypoxia-inducible factor-2α (HIF-2α) and its target genes, erythropoietin (EPO) and endothelin-1, causes polycythemia and pulmonary hypertension in patients with Chuvash polycythemia who are homozygous for the R200W mutation in the von Hippel Lindau (VHL) gene and in a murine mouse model of Chuvash polycythemia that bears the same homozygous VhlR200W mutation. Moreover, the aged VhlR200W mice developed pulmonary fibrosis, most likely due to the increased expression of Cxcl-12, another Hif-2α target. Patients with mutations in iron regulatory protein 1 (IRP1) also develop polycythemia, and Irp1-knockout (Irp1-KO) mice exhibit polycythemia, pulmonary hypertension, and cardiac fibrosis attributable to translational derepression of Hif-2α, and the resultant high expression of the Hif-2α targets EPO, endothelin-1, and Cxcl-12. In this study, we inactivated Hif-2α with the second-generation allosteric HIF-2α inhibitor MK-6482 in VhlR200W, Irp1-KO, and double-mutant VhlR200W;Irp1-KO mice. MK-6482 treatment decreased EPO production and reversed polycythemia in all 3 mouse models. Drug treatment also decreased right ventricular pressure and mitigated pulmonary hypertension in VhlR200W, Irp1-KO, and VhlR200W;Irp1-KO mice to near normal wild-type levels and normalized the movement of the cardiac interventricular septum in VhlR200Wmice. MK-6482 treatment reduced the increased expression of Cxcl-12, which, in association with CXCR4, mediates fibrocyte influx into the lungs, potentially causing pulmonary fibrosis. Our results suggest that oral intake of MK-6482 could represent a new approach to treatment of patients with polycythemia, pulmonary hypertension, pulmonary fibrosis, and complications caused by elevated expression of HIF-2α.

Intraperitoneal injection of Desferal\xae alleviated the age-related bone loss and senescence of bone marrow stromal cells in rats

BackgroundAge-related bone loss plays a vital role in the development of osteoporosis and osteoporotic fracture. Bone marrow stromal cell (BMSC) senescence is highly associated with osteoporosis and limits the application of BMSCs in regenerative medicine. Hypoxia is an essential component for maintaining the normal physiology of BMSCs. We have reported that activation of hypoxia-induced factor by deletion of von Hippel-Lindau gene in osteochondral progenitor cells protected mice from aging-induced bone loss. However, whether pharmacologically manipulation of hypoxic niche would attenuate age-related bone loss and dysfunction of BMSCs is not well understood.MethodsTwelve-month-old Sprague-Dawley rats were used as an aged model and were intraperitoneally injected with Desferal® (20, 60 mg/kg weight or vehicle), three times a week for a continuous 8-week period. Two-month-old young rats were set as a reference. After 8 weeks, micro-CT and HE staining were performed to determine the effect of Desferal® on bone loss. In order to investigate the effects of Desferal® on BMSC senescence, 12-month-old rats were treated with high-dose Desferal® (60 mg/kg weight) daily for 10 days. BMSCs were isolated and evaluated using CCK-8 assay, colony-forming cell assay, cell differentiation assay, laser confocal for reactive oxygen species (ROS) level, senescence-associated β-galactosidase (SA-β-gal) staining, and molecular expression test for stemness/senescence-associated genes.ResultsMicro-CT and HE staining showed that high-dose Desferal® significantly prevented bone loss in aged rats. Compared with vehicle group, the ex vivo experiments showed that short-term Desferal® administration could promote the potential of BMSC growth (proliferation and colony formation ability) and improve the rebalance of osteogenic and adipogenic differentiation, as well as rejuvenate senescent BMSCs (ROS level and SA-β-gal staining) and revise the expression of stemness/senescence-associated genes. The potential of BMSCs from 12M-H-Desferal® group at least partly revised to the level close to 2-month-old group.ConclusionsThe current study suggested that Desferal®, an iron-chelating agent, could alleviate age-related bone loss in middle-aged rats. Meanwhile, we found that short-term intraperitoneal injection of Desferal® partly rejuvenate BMSCs from aged rats. Overall, we demonstrated a novel role of Desferal® in rejuvenating aged BMSCs and preventing age-related bone loss.

Identification and validation of the clinical roles of the VHL-related LncRNAs in clear cell renal cell carcinoma.

Accumulating evidence suggests that lncRNAs (long non-coding RNAs) function as oncogenes or tumor suppressor genes in ccRCC (clear cell renal cell carcinoma). Although VHL (Von Hippel-Lindau) gene inactivation is by far the most common carcinogenic driving event in ccRCC, the roles of VHL-related lncRNAs in ccRCC remain unknown. In this study, using RNA-seq and clinical data in TCGA-KIRC (the Cancer Genome Atlas-Kidney Renal Clear Cell Carcinoma), we identified VHL-related lncRNAs through WGCNA (Weighted Gene Co-expression Network Analysis), correlation analysis and catRAPID algorithm, and explored their clinical characteristics in ccRCC. Results showed that 10 lncRNAs (AC112220.2, AL391121.1, USP46-AS1, AL450326.1, MID1IP1-AS1, SUCLG2-AS1, RAP2C-AS1, FGD5-AS1, AC018647.2 and AC015922.2) were identified as VHL-related lncRNAs, and they were down-regulated in ccRCC tissues. Survival analysis results indicated that high expression groups of AC112220.2, AL391121.1, USP46-AS1, AL450326.1, SUCLG2-AS1, RAP2C-AS1, FGD5-AS1, AC018647.2 and AC015922.2 had significantly longer OS (Overall Survival) than their respective low expression groups. Meanwhile high AC112220.2, USP46-AS1, AL450326.1, SUCLG2-AS1, FGD5-AS1, AC018647.2 and AC015922.2 expression groups had remarkably longer DFS (Disease Free Survival) than their respective low expression groups. Besides, FGD5-AS1 and AL391121.1 expression were decreased in VHL mutant tissues compared with VHL non-mutant tissues. Moreover, high expression group of FGD5-AS1 had significantly longer OS and DFS than their respective low expression groups in VHL mutant ccRCC. In addition, we found that DNA hypermethylation may also play an important role in decreased FGD5-AS1 expression. Furthermore, we validated the expression of FGD5-AS1 in VHL mutant and non-mutant ccRCC tissues and cell lines. In conclusion, our results demonstrated that lncRNA FGD5-AS1 was significantly associated with VHL and can serve as a novel biomarker of ccRCC.

Deficiency of the X-inactivation escaping gene KDM5C in clear cell renal cell carcinoma promotes tumorigenicity by reprogramming glycogen metabolism and inhibiting ferroptosis.

Background: Clear cell renal cell carcinoma (ccRCC) is characterized by glycogen-laden, unexplained male predominance, and frequent mutations in the Von Hippel-Lindau (VHL) gene and histone modifier genes. Besides, poor survival rates of ccRCC patients seem to be associated with up-regulation of the pentose phosphate pathway (PPP). However, the mechanism underlying these features remains unclear.Methods: Whole exome sequencing was used to identify the gene mutation that implicated in the rewired glucose metabolism. RNA-seq analyses were performed to evaluate the function of KDM5C in ccRCC. Furthermore, heavy isotope tracer analysis and metabolites quantification assays were used to study how KDM5C affects intracellular metabolic flux. To provide more in vivo evidence, we generated the Kdm5c-/- mice by CRISPR-Cas9 mediated gene knockout and performed the xenografts with KDM5C overexpressing or depleted cell lines.Results: A histone demethylase gene KDM5C, which can escape from X-inactivation and is predominantly mutated in male ccRCC patients, was identified to harbor the frameshift mutation in the ccRCC cell line with the highest glycogen level, while the restoration of KDM5C significantly reduced the glycogen level. Transcriptome and metabolomic analysis linked KDM5C to metabolism-related biological processes. KDM5C specifically regulated the expression of several hypoxia-inducible factor (HIF)-related genes and Glucose-6-phosphate dehydrogenase (G6PD) that were involved in glycogenesis/glycogenolysis and PPP, respectively, mainly through the histone demethylase activity of KDM5C. Depletion of KDM5C increased the production of glycogen, which was then directed to glycogenolysis to generate glucose-6-phosphate (G6P) and subsequently PPP to produce nicotinamide adenine dinucleotide phosphate hydride (NADPH) and glutathione (GSH), thus conferring cells resistance to reactive oxygen species (ROS) and ferroptosis. KDM5C re-expression suppressed the glucose flux through PPP and re-sensitized cancer cells to ferroptosis. Notably, Kdm5c-knockout mice kidney tissues exhibited elevated glycogen level, reduced lipid peroxidation and displayed a transformation of renal cysts into hyperplastic lesions, implying a cancer-protective benefit of ferroptosis. Furthermore, KDM5C deficiency predicted the poor prognosis, and clinically relevant KDM5C mutants failed to suppress glycogen accumulation and promoted ferroptosis as wild type.Conclusion: This work revealed that a histone modifier gene inactive mutation reprogramed glycogen metabolism and helped to explain the long-standing puzzle of male predominance in human cancer. In addition, our findings may suggest the therapeutic value of targeting glycogen metabolism in ccRCC.

Loss of Function of von Hippel-Lindau Trigger Lipocalin 2-Dependent Inflammatory Responses in Cultured and Primary Renal Tubular Cells.

Previous studies have shown that mutations in the tumor suppressor gene von Hippel-Lindau (VHL) can result in the overproduction of reactive oxygen species (ROS) and chronic inflammation and are a significant predisposing factor for the development of clear-cell renal cell carcinoma (ccRCC). To study VHL's role in ccRCC formation, we previously developed a novel conditional knockout mouse model that mimicked the features of kidney inflammation and fibrosis that lead to cyst formation and hyperplasia. However, due to VHL's complex cellular functions, the mechanism of this phenomenon remains unclear. Here, we used the HK-2 cells and mouse primary renal tubule cells (mRTCs) carrying VHL mutations as models to study the effects and underlying molecular mechanisms of ROS accumulation. We also studied the role of lipocalin 2 (LCN2) in regulating macrophage recruitment by HK-2 cells. We measured the level of ROS in HK-2 cells in the presence or absence of LCN2 knockdown and found that the VHL mutation caused ROS overproduction, but an LCN2 knockdown could attenuate the process. VHL was also found to mediate the in vitro and in vivo expression and secretion of LCN2. Thus, VHL likely affects ROS production in an LCN2-dependent manner. Our findings also suggest that LCN2 sensitizes the inflammatory response of HK-2 cells and the chemotactic abilities of macrophage RAW264.7 cells. By demonstrating that the loss of function of von Hippel-Lindau triggers lipocalin 2-dependent inflammatory responses in cultured and primary renal tubular cells, our results offer novel insights into a potential therapeutic approach for interfering with the development of ccRCC.

Phthisis bulbi and retinal capillary haemangioma associated with papilloedema due to recurrent bilateral cerebellar haemangioblastomas – A case of Von Hippel Lindau syndrome

Von Hippel Lindau (VHL) syndrome is an autosomal dominant condition caused by mutation of VHL gene. It is characterized by vascular tumors of eye, central nervous system, visceral cysts and tumors. Here we report a case of VHL in a young male patient who presented with phthisis bulbi in the right eye and retinal capillary haemangioma in the left eye. Systemic features of raised intracranial pressure was present along with bilateral abduction deficit and florid disc edema in the left eye. There was a polypoidal reddish mass in the inferotemporal quadrant, with a dilated draining vein, indicating retinal capillary hemangioma. The phthisical right eye was speculated to result from untreated retinal capillary hemangioma, culminating in exudative retinal detachment. Urinary Vanillyl Mandelic Acid suggested pheochromocytoma. Imaging modalities revealed multiple cerebellar hemangioblastomas, pheochromocytoma and pancreatic cysts. Screening of family members revealed no evidence of systemic manifestations, suggesting the possibility of sporadic mutation. Retinal capillary hemangioma is the commonest and earliest manifestation of VHL. The ocular complications include intraretinal exudation and exudative retinal detachment, secondary angiomatosis, epiretinal membrane, vitreous hemorrhage, retinal neovascularization, tractional retinal detachment and macular ectopia. Our case report emphasizes that retinal examination in patients with suspected VHL is not only a screening tool for the presence of capillary hemangioma but also for regular monitoring and prevention of vision threatening complications. Keywords: Von Hippel Lindau (VHL) syndrome, Retinal capillary hemangioma, Cerebellar hemangioblastomas, Pheochromocytoma.

VHL gene methylation contributes to excessive erythrocytosis in chronic mountain sickness rat model by upregulating the HIF-2α/EPO pathway

AimsHypoxia-inducible factors (HIFs) play important roles in the pathogenesis of erythrocytosis in chronic mountain sickness (CMS). von Hippel-Lindau (VHL) is a key regulator of hypoxia that can direct the poly-ubiquitylation and degradation of HIFs. Epigenetic mechanisms are believed to contribute toward adaption to chronic hypoxia. Here, we investigated the contribution and mechanism of VHL methylation in rats with erythrocytosis in CMS. Main methodsThe methylation status of VHL was measured via bisulfite sequencing PCR, while VHL, DNMT1, DNMT3α, and DNMT3β expression were assessed using real-time reverse transcription PCR and western blotting. HIF-2α and EPO expression levels in bone marrow were determined via immunohistochemical staining, and erythroid hyperplasia in bone marrow sections were observed with hematoxylin and eosin staining. Key findingsWe found that chronic hypoxia triggered erythroid hyperplasia in the bone marrow and increased the quantity of peripheral red blood cells in CMS rats. Chronic hypoxia significantly induced methylation at the CpG site in the VHL promoter, decreased VHL expression, and increased HIF-2α and EPO expression. Chronic hypoxia increased DNMT3α and DNMT3β expression, consistent with the decrease in VHL expression. The DNA methyltransferase inhibitor 5-azacytidine reduced chronic hypoxia-induced erythroid proliferation in the bone marrow of rats with CMS by suppressing VHL methylation and DNMTs expression. SignificanceOur study suggests that VHL methylation contributes toward excessive erythrocytosis in CMS by upregulating the HIF-2α/EPO pathway in the bone marrow of rats. We demonstrated that the DNMT inhibitor 5-azacytidine can attenuate erythroid hyperplasia in the bone marrow by demethylating the VHL promoter.

Ultrasound Microbubble-Mediated VHL Regulates the Biological Behavior of Ovarian Cancer Cells

According to the literature, the von Hippel–Lindau (VHL) gene has a certain correlation with ovarian cancer. In this study, we investigated the effect and mechanism of ultrasound microbubble-mediated VHL on the biological function of ovarian cancer cells. Non-targeting lipid microbubbles and targeted lipid microbubbles were prepared. OVCAR-3 cells were treated with VHL mediated by ultrasound and microbubbles alone or together. Expressions of VHL, Akt, epithelial–mesenchymal-transition-related proteins and apoptosis-related proteins were detected by Western blot and quantitative real-time polymerase chain reaction as needed. The effect of ultrasound microbubble-mediated VHL on the proliferation, apoptosis, cell cycle, migration and invasion of OVCAR-3 cells was examined by Cell Counting Kit-8, flow cytometry, wound-healing assay and Transwell. Compared with other treatment methods, ultrasound microbubble mediation enhanced VHL expression in OVCAR-3 cells. Overexpression of liposome-mediated VHL inhibited the proliferation and migration; caused cell-cycle arrest; promoted apoptosis: downregulated the expressions of MMP2, MMP9, E-cadherin, Akt and Bcl-2; and upregulated the expressions of VHL and BCL2-associated X protein (BAX) in OVCAR-3 cells. The effect of microbubble-treated VHL was similar to liposome-mediated regulation, while ultrasound treatment enhanced the effect of VHL on OVCAR-3 cells. More interestingly, ultrasound microbubble-mediated VHL had the most obvious regulatory effect on OVCAR-3 cells. Ultrasound microbubble technology increases the transfection efficiency of VHL into OVCAR-3 cells and enhances the effect of VHL gene on the biological function of OVCAR-3 cells.

Phlebotomy-Induced Iron Deficiency Increases the Expression of Prothrombotic Genes

Thrombosis is the major cause of morbidity and mortality in Chuvash erythrocytosis (CE), caused by a hypomorphic R200W mutation of the Von Hippel-Lindau (VHL) gene, a negative regulator of hypoxia inducible factors (HIFs). This mutation augments HIF activity even in normoxia. High hematocrit associated with hyperviscosity has been considered a risk factor for thrombosis suggesting that maintaining normal hematocrit may prevent thrombosis. However, our prospective controlled study reported that phlebotomies, but not the high hematocrit, are associated with increased risk of thrombosis (PMID 289208) by a mechanism that remains to be elucidated. Phlebotomies lead to iron deficiency. We explored the potential role of iron deficiency as a cause of thrombosis. Iron deficiency induces HIF activity by inhibiting prolyl hydroxylase domain 2 (PHD2), an inhibitor of HIFs that requires iron as a co-factor (PMID 18066546). The expression of certain prothrombotic genes regulated by HIFs is modestly increased in CE mononuclear cells (PMID 23993337). We hypothesized that further augmentation of already high HIF activity by iron deficiency might further increase expression of prothrombotic genes leading to increased risk of thrombosis in phlebotomized subjects. We reported that, in polycythemia vera (PV) and essential thrombocythemia (ET), the pattern of increase in prothrombotic and inflammatory gene expression differs between granulocytes and platelets (PMID: 32203583), suggesting cell-specific contributions to thrombosis in these disorders. We analyzed the whole transcriptome of the platelets of 10 CE patients, 6 with iron deficiency (ferritin &amp;lt;20 ng/ml) and 4 with normal ferritin: 2,412 genes were upregulated and 670 were downregulated (p &amp;lt;0.05 and Log2 fold change &amp;gt;1). Dysregulated genes as analyzed by Ingenuity Pathway Analysis (Qiagen) were associated with platelet binding, hemostasis and thrombus signaling and decreased bleeding time. To explore our hypothesis further, we quantitated the mRNA of these HIF-regulated prothrombotic genes: THBS1 (thrombospondin 1), SERPINE1 (plasminogen activator inhibitor-1 [PAI-1]), ITGA2B (integrin alpha-IIb), PTGS2 (prostaglandin-endoperoxide synthase 2), SELP (P-selectin), PDGFA (platelet derived growth factor subunit A), and ITGB3 (integrin beta-3). We analyzed granulocytes from 16 CE subjects (8 iron deficient) and platelets from 12 CE subjects (7 iron deficient). In platelets, THBS1, SELP, SERPINE1, and PDGFA mRNA levels were higher in iron deficient CE subjects than those with normal ferritin (p=0.015-0.088). In all CE subjects, the mRNA levels of these four genes correlated inversely with ferritin (figure 1). PTGS2 (known to be down regulated in thrombosis) was down regulated in iron deficient CE patients and correlated positively with ferritin. ITGB3 and ITGA2B mRNA levels were not different between the two groups. In granulocytes, SELP mRNA was augmented in CE patients with iron deficiency and both SELP and ITGB3 mRNA levels correlated inversely with ferritin. We did not find a difference in expression of KLF2, a regulator of thrombotic genes, in iron deficient versus iron sufficient CE patients; see this meeting Song J et al. We then tested our hypothesis of augmentation of thrombosis risk by iron deficiency in granulocytes from 50 PV and ET patients (9 with iron deficiency) and platelets from 41 patients (5 with iron deficiency). In granulocytes, THBS1, SELP, and IRAK1 (Interleukin 1 Receptor Associated Kinase 1) mRNA levels were higher in patients with iron deficiency, and IRAK1, THBS1, and SERPINE1 mRNA levels correlated inversely with ferritin. In platelets, THBS1, and SERPINE1 mRNA were higher in patients with iron deficiency and SELP, THBS1, and SERPINE1 mRNA levels correlated inversely with ferritin. JAK2V617F allele burden also correlated inversely with ferritin. Our study demonstrates that iron deficiency is associated with increased expression of HIF-regulated prothrombotic genes in CE platelets and granulocytes in a pattern that differs between these two cell types. We also report increased expression of prothrombotic genes in PV and ET patients with iron deficiency. These results underline the potential danger of phlebotomies in attempts to control high hematocrit. We caution against indiscriminate use of therapeutic phlebotomy for treatment of patients with PV and other erythrocytoses. VG &amp;JTP contributed equally Figure Disclosures Gordeuk: Ironwood: Research Funding; Novartis: Consultancy; CSL Behring: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Imara: Research Funding.

Mass Spectrometry-Based Metabolic Fingerprinting Contributes to Unveil the Role of RSUME in Renal Cell Carcinoma Cell Metabolism.

Clear cell renal cell carcinoma (ccRCC) is a heterogeneous disease with 50-80% patients exhibiting mutations in the von Hippel-Lindau (VHL) gene. RSUME (RWD domain (termed after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases)-containing protein small ubiquitin-related modifier (SUMO) enhancer) acts as a negative regulator of VHL function in normoxia. A discovery-based metabolomics approach was developed by means of ultraperformance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (MS) for fingerprinting the endometabolome of a human ccRCC cell line 786-O and three other transformed cell systems (n = 102) with different expressions of RSUME and VHL. Cross-validated orthogonal projection to latent structures discriminant analysis models were built on positive, negative, and a combination of positive- and negative-ion mode MS data sets. Discriminant feature panels selected by an iterative multivariate classification allowed differentiating cells with different expressions of RSUME and VHL. Fifteen identified discriminant metabolites with level 1, including glutathione, butyrylcarnitine, and acetylcarnitine, contributed to understand the role of RSUME in ccRCC. Altered pathways associated with the RSUME expression were validated by biological and bioinformatics analyses. Combined results showed that in the absence of VHL, RSUME is involved in the downregulation of the antioxidant defense system, whereas in the presence of VHL, it acts in rerouting energy-related pathways, negatively modulating the lipid utilization, and positively modulating the fatty acid synthesis, which may promote deposition in droplets.

The von Hippel-Lindau Tumor Suppressor Gene: Implications and Therapeutic Opportunities.

The discovery of the von Hippel-Lindau (VHL) gene marked a milestone in our understanding of clear cell renal cell carcinoma (ccRCC) pathogenesis. VHL inactivation is not only a defining feature of ccRCC, but also the initiating event. Herein, we discuss canonical and noncanonical pVHL functions, as well as breakthroughs shaping our understanding of ccRCC evolution and evolutionary subtypes. We conclude by presenting evolving strategies to therapeutically exploit effector mechanisms downstream of pVHL.