Introducing the BHD and Folliculin Research Symposium 2023
Flash Talks
Presenting Author: Evelyn Lynn, University College Dublin, Ireland
Presenting Author Position: Specialist Registrar in Respiratory Medicine & M.D. Candidate
Abstract Title: Automated cyst burden score as a measure of lung disease in Birt-Hogg-Dube Syndrome.
Authors and Affiliations: Evelyn Lynn1,2, Yasuhito Sekimoto2, Brian Gaffney3, Maitreyi Penugonda1, Joseph Morrow3, David J. Murphy,2,3. Jonathan D. Dodd3, Michael P. Keane1,2, Cormac McCarthy1 1Department of Respiratory Medicine, St. Vincent’s University Hospital, Elm Park, Dublin 4, Ireland, 2Conway Institute, University College Dublin, 3Department of Radiology, St. Vincents University Hospital.
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Background: Birt-Hogg-Dube Syndrome (BHDS) is an autosomal dominant disease with a reported population prevalence is 2 per million. We describe the characteristics of our cohort and assess the role of automated cyst burden score in BHDS, examining correlation with lung function. Methods: Clinical features, genetic mutations and pulmonary function was collated. SyngoVia post-processing software determined CT cyst scores using imaging slices of 3mm and a cut off range of – 900 to -950 Hounsfield Units. Low attenuation cysts were clustered by volume and divided into 4 classes; Class 1: 2-10 mm3, Class 2: 10-50 mm3, Class 3: 50-150 mm3, Class 4: >150 mm3. Total lung volume by each class was then calculated. Cyst scores were correlated with pulmonary function. Results: Thirty-one patients from 15 families were identified. Median age was 53.82 years (SD+/-15.5 years). Fifty-five percent (n=17) were female. Six unique genetic mutations were identified, C.17_21delCTCTC (45%, n=8) the most prevalent. Forty-five percent (n=14) had history of pneumothorax, 35% (n=9) had fibrofolliculomas and 6% (n=2) had renal tumours. Mean FEV1 and DLCO were 91% (SD+/-17.3%) and 80% (SD+/-13%) respectively. Mean cyst burden score was 2% of total lung volume (range 0.1-5.4%). Cyst burden did not correlate with FEV1% (R2=0.079,p=0.429) or DLCO (R2=0.023,p=0.676). Patients with a history of pneumothorax were more likely to have higher cyst burden score independent of lung function (mean=2.37% vs 0.9%,p=0.08). Conclusion: Our data suggests the prevalence of BHDS is 6.2 cases per million in Ireland. A history of pneumothorax correlated with higher cyst burden but otherwise cyst burden did not correlate with pulmonary function. We aim to further explore the value of cyst burden scores in BHDS and other diffuse cystic lung diseases.
References: Muller ME, Daccord C, Taffé P, Lazor R. Prevalence of Birt-Hogg-Dubé Syndrome Determined Through Epidemiological Data on Spontaneous Pneumothorax and Bayes Theorem. Front Med (Lausanne). 2021 Apr 27;8:631168. doi: 10.3389/fmed.2021.631168. PMID: 33987191; PMCID: PMC8111214.
Presenting Author: Jennifer Heritz, SUNY Upstate Medical University, Syracuse, NY, USA
Presenting Author Position: PhD student
Abstract Title: SUMOylation of FLCN is required for its tumor suppressor activity.
Authors and Affiliations: Jennifer Heritz1,2,3, Sara Cayaban1,3, Gennady Bratslavsky1,2,3, Mark Woodford1,2,3, Mehdi Mollapour1,2,3 1 Department of Urology, SUNY Upstate Medical University, Syracuse, NY, USA. 2 Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA. 3 Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY, USA.
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Germline mutation of the tumor suppressor FLCN (Folliculin) is associated with the rare inherited cancer syndrome Birt-Hogg-Dubé (BHD). BHD patients develop kidney tumors, pulmonary cysts, and benign skin tumors (fibrofolliculomas). Canonically, FLCN participates in the regulation of mTOR through its role as a GTPase-activating protein for the GTPase RagC. However, the precise mechanism of FLCN tumor suppressive function is unknown. Notably, it was previously shown that loss of FLCN leads to increased lactate dehydrogenase A (LDHA) activity. Our published work revealed that FLCN binds to and inhibits LDHA activity in normal cells to regulate the Warburg effect. Although mutations in FLCN generally lead to instability of the protein, the functional impact of these mutations, which are distributed throughout its coding sequence, remain elusive. Here, we engineered and exogenously expressed 19 pathogenic mutations of FLCN in the FLCN-null UOK257 cell line, which is established from a patient with BHD syndrome. The protein expression of these FLCN mutants were generally divided into two categories: stable and unstable. Treatment of cells expressing the unstable FLCN mutants with the proteasome inhibitor bortezomib stabilized the FLCN mutants. This stabilization restored FLCN interaction with LDHA and consequent inhibition of the enzyme’s activity. Furthermore, we have discovered a lysine site within FLCN that facilitates FLCN binding to LDHA, which is subject to SUMOylation. Although pathogenic mutation of this lysine site does not impact the stability of FLCN, the lack of SUMOylation abrogates FLCN binding to LDHA and consequently leads to LDHA hyperactivity. Taken together, our data presented here provides a complex landscape of the impact of pathogenic mutations towards FLCN function and provides a strategy to restore the tumor suppressive activity of FLCN.
References: 1. Linehan WM, et al. The Metabolic Basis of Kidney Cancer. Cancer Discov, (2019). 2. Nahorski MS, et al. Birt Hogg-Dube syndrome associated FLCN mutations disrupt protein stability. Human mutation 32, 921-929 (2011). 3. Woodford MR, et al. The tumor suppressor folliculin inhibits lactate dehydrogenase A and regulates the Warburg effect. Nat Struct Mol Biol 28, 662-670 (2021).
Presenting Author: Melina Michael, University College London, UK
Presenting Author Position: Post Doc
Abstract Title: Unlocking the Potential of Tumour-Primed NK Cells to Combat Renal Cell Cancer
Authors and Affiliations: Professor Mark Lowdell University College London, UK
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Renal cell cancer (RCC) is a prevalent malignancy arising from renal tubular epithelial cells in the kidney, accounting for over 90% of primary kidney tumours and exhibiting a high mortality rate. Natural Killer (NK) cells, as innate lymphoid cells, play a crucial role in host defence against infections and cancers. Recently, NK cells have garnered considerable attention in cancer immunotherapy due to their innate effector functions and their capacity for adaptive immune processes, such as priming and long-term memory-like responses. Tumour-primed NK cells (TpNK) have been pre-exposed to tumour cells in vitro, resulting in their activation and acquisition of memory-like features, including enhanced lytic function upon re-stimulation. Notably, TpNK cells induced by INB16, an acute lymphoblastic leukaemia cell line, have demonstrated augmented lytic functions in both in vitro and in vivo settings, encompassing various haematological and solid tumour models. A clinical trial is currently underway, investigating the efficacy of TpNK cells in myelodysplastic syndrome, utilizing INKmuneTM, a proprietary cell drug administered intravenously to prime autologous NK cells in vivo. Here, we show enhanced lytic activity of TpNK compared to resting NKs from the same healthy donors in the RCC cell line, 786-O. Ongoing work will determine if NK cells of patients with RCC can be enhanced by INKmuneTM, and how to predict which patients will most likely benefit, with the aim to support a UK clinical trial application for TpNK in RCC.
Presenting Author: Embolo Enyegue Elisée Libert, Institute of Medical Research and Medicinal Plant Studies (IMPM), Cameroon
Presenting Author Position: Research Fellow
Abstract Title: Investigation of active peptides from natural products that induce ER-stress-mediated apoptosis in cancer as potential therapeutics for kidney cancer, common in some genetic diseases: An in silico approach.
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Background: The endoplasmic reticulum (ER) stress initiates unfolded protein response (UPR) to re-establish ER homeostasis as an adaptive pathway in cancer. However, persistent ER stress triggers the apoptotic pathway. The term “kidney cancer” (KC) actually refers to a number of distinct cancers, each of which has its own distinctive histology, genetic alterations, clinical course, and therapeutic response. Apart from the usual socio-economic factors generally implicated in the occurrence of cancer; kidney cancer is generally observed in a number of genetic diseases such as: Von hippel-lindau syndrome (VHL), Tuberous sclerosis complex (TSC), Birt hogg dube syndrome (BHD), Hereditary papillary renal cell carcinoma, Hereditary leiomyomatosis, Hereditary paranglioma and pheochromocytoma syndrome, Downden’s syndrome. Historically, patients with renal tumors underwent the same surgical procedures, and received similar drug treatments, none of which worked, for the treatment of KC. The available systemic therapies did not increase survival for patients with advanced disease, although there would occasionally be a response. Despite significant improvements in cancer detection and treatment, one of the most difficult aspects of the field is still disease management especially for those living with genetic diseases. Methods: The purpose of this research was to generate peptides from natural in sillico that might be used as kidney cancer therapeutics. Target prediction, protein-protein interaction, and protein-peptide molecular docking have all been used as calculation techniques. Results: The network of critical KC gene consist of C3AR1, CSNK2A2, ACE, DPP4, CAPN1, FPR2, HLA-A and MMP2 together with predicted kinases such as RPS6KA5, MAPK14, CSNK2A1, PRKCD, CDK1 and HIPK2in addition to transcription factors such as IRF8, TCF3, ERG, CREB1, EZH2, SPI1, IRF1 and SUZ12. The identified molecular target of isolated peptides HLA class I histocompatibility antigen A-3, Lipoxin A4, Dipeptidyl peptidase IV, Angiotensinconverting enzyme, Cyclooxygenase-2, C3a anaphylatoxin chemotactic receptor, Melanocortin receptor 4, Neurotensin receptor 1, Mu opioid receptor, Delta opioid receptor and Calpain 1. Conclusion: Overall, the results showed that GVSK, PGP, WQR, YGGF and IF peptides are promising candidates for further study. Future work would be needed to test the therapeutic properties of these hydrolysate peptides by in vitro and in vivo approaches.
References: Kim, C., & Kim, B. (2018). Anti-Cancer Natural Products and Their Bioactive Compounds Inducing ER Stress-Mediated Apoptosis: A Review. Nutrients, 10(8), 1021. https://doi.org/10.3390/nu10081021 Kurrikoff, K., Aphkhazava, D., & Langel, Ü. (2019). The future of peptides in cancer treatment. Current Opinion in Pharmacology, 47, 27–32. https://doi.org/10.1016/j.coph.2019.01.008 Latif, F., Tory, K., Gnarra, J., Yao, M., Duh, F. M., Orcutt, M. L., Stackhouse, T., Kuzmin, I., Modi, W., & Geil, L. (1993). Identification of the von Hippel-Lindau disease tumor suppressor gene. Science (New York, N.Y.), 260(5112), 1317–1320. https://doi.org/10.1126/science.8493574 Linehan, W. M., & Ricketts, C. J. (2013). The metabolic basis of kidney cancer. Seminars in Cancer Biology, 23(1), 46–55. https://doi.org/10.1016/j.semcancer.2012.06.002 Nickerson, M. L., Warren, M. B., Toro, J. R., Matrosova, V., Glenn, G., Turner, M. L., Duray, P., Merino, M., Choyke, P., Pavlovich, C. P., Sharma, N., Walther, M., Munroe, D., Hill, R., Maher, E., Greenberg, C., Lerman, M. I., Linehan, W. M., Zbar, B., & Schmidt, L. S. (2002). Mutations in a novel gene lead to kidney tumors, lung wall defects, and benign tumors of the hair follicle in patients with the Birt-Hogg-Dubé syndrome. Cancer Cell, 2(2), 157–164. https://doi.org/10.1016/s1535-6108(02)00104-6.
Presenting Author: Ye Yang, National Institute of Health, USA
Presenting Author Position: Research Fellow
Abstract Title: Metabolic Characterization of Birt-Hogg-Dubé Syndrome Renal Tumor Cells and Tissues Using Stable Isotope-Resolved Metabolomics
Authors and Affiliations: Ye Yang 1,2, Daniel R. Crooks 1,2, Christopher J. Ricketts 1, Richard M. Higashi 3, Teresa W.-M. Fan 3, Andrew Lane 3, Laura S. Schmidt 1,4, Nunziata Maio 5, Youfeng Yang 1, Cathy D. Vocke 1, W. Marston Linehan 1 1 Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 2 Clinical Cancer Metabolism Facility, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 3 Department of Toxicology and Cancer Biology, Markey Cancer Center, University of Kentucky, Lexington, KY, USA 4 Basic Science Program, Frederick National Laboratory for Cancer Research, Frederick, MD, USA 5 Molecular Medicine Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD 20892 USA.
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Background Birt-Hogg-Dubé syndrome (BHD) is caused by germline mutations in the FLCN gene, and patients are at risk of developing bilateral, multifocal renal tumors [1]. While multiple potential roles of FLCN have been identified, such as AKT-mTOR pathway signaling, AMPK signaling, and TFE3/TFEB transcriptional activation [2], there are few studies that have investigated the activity of central metabolic pathways in BHD human renal tumors and cell lines. Methods In the current study we utilized Stable Isotope Resolved Metabolomics (SIRM) to investigate and characterize the altered metabolic pathways in patient-derived BHD renal tumor cell lines (UOK257) [3] and renal tumor slices obtained intra-operatively from patients undergoing surgery at the NIH Clinical Center. NMR as well as ultra-high-resolution mass spectrometry were used to analyze the polar/non-polar metabolites extracted from BHD renal tumor cells and tissues. Results and Conclusions Our data revealed that the BHD tumor tissues exhibit enhanced glucose oxidation and reduced glutamine uptake relative to renal cortex tissues. Using 13C6-glucose as the tracer, we found increased citrate (m+2)/pyruvate (m+3) in BHD tumor tissues, which suggested enhanced pyruvate dehydrogenase (PDH) activity relative to renal cortex tissues. This was consistent with the gene expression analysis. Moreover, western blot analysis demonstrated that the respiratory chain was also upregulated in the BHD tumors. Treatment of UOK257 cells with respiratory chain inhibitor metformin inhibited cell growth. 13C6-glucose tracer experiments demonstrated that the oxidation of glucose through PDH pathway was inhibited. Whereas 13C5,15N2-glutamine tracer experiments showed that while the oxidative glutamine metabolism was inhibited, the reductive carboxylation of glutamine was stimulated with metformin treatment of UOK257 cells . Metformin also decreased the incorporation of glucose derived 13C into lipid acyl chain in UOK257 cells. These findings provide a potential foundation for the development of therapeutic approaches for treatment and/or prevention of BHD renal cancer.
References: 1. Vocke, C.D., Yang, Y., Pavlovich, C.P., Schmidt, L.S., Nickerson, M.L., Torres-Cabala, C.A., Merino, M.J., Walther, M.M., Zbar, B., Linehan, W.M., High frequency of somatic frameshift BHD gene mutations in Birt-Hogg-Dube-associated renal tumors. J. Natl. Cancer Inst. 2005; 97: 931–935. 2. Schmidt, L.S., Linehan, W.M., FLCN: The causative gene for Birt-Hogg-Dubé syndrome. Gene. 2018; 640: 28-42. 3. Yang, Y., Padilla-Nash, H. M., Vira, M. A., Abu-Asab, M. S., Val, D., Worrell, R., Tsokos, M., Merino, M. J., Pavlovich, C. P., Ried, T., Linehan, W. M., & Vocke, C. D. (2008). The UOK 257 cell line: a novel model for studies of the human Birt-Hogg-Dubé gene pathway. Cancer genetics and cytogenetics, 180(2), 100–109. Funded in part by FNLCR Contract HHSN261201500003I.