Driving research, providing support and improving outcomes for patients and their families affected by rare conditions

Introducing the BHD and Folliculin Research Symposium 2023

Abstract Talks

Presenting Author: Dr. Nicola Alesi, Brigham & Women’s Hospital, USA

Abstract Title: TFEB hyperactivation is the missing link between Tuberous Sclerosis Complex and Birt-Hogg-Dube.

Abstract Text

mTORC1 phosphorylates the Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, at Ser211. In Alesi et al. Nature Communications, 2021 we found that lysosomal biogenesis is increased in tumors of TSC patients, kidneys from Tsc2+/− mice, and TSC1/2-deficient cells via a TFEB-dependent mechanism. In TSC1/2-deficient cells, TFEB is paradoxically hypo-phosphorylated at the mTORC1-dependent site S211, resulting in nuclear localization and hyperactivation. Importantly, overexpression of folliculin (FLCN), a GTPase activating protein for RAGC, increases TFEB phosphorylation at the mTORC1 sites in TSC2-deficient cells, suggesting cooperation between TSC2 and FLCN in the regulation of TFEB. In unpublished work, we found that TFEB is the primary driver of kidney tumorigenesis in two novel mouse models of TSC, similarly to what has been observed in Napolitano et al. Nature, 2020 in a kidney-specific mouse model of Birt-Hogg-Dube (BHD). Specifically, we developed a mouse model in which TFEB is knocked out in the kidney (KspCre; Tsc2fl/fl), resulting in cystic kidney disease and a 10-fold increase in kidneys/body weight ratio. Double knockout of TSC2 and TFEB (KspCre; Tsc2fl/fl Tfebfl/fl) completely abolished kidney cysts, normalized the kidney/body weight ratio, renal function, and extended lifespan. We also studied an inducible total body model of TSC (Cagg-CreERT2; Tsc2fl/fl) in which Tamoxifen is administered at day P1-3. These mice have decreased body size (~50%) and cystic kidneys. TFEB knockdown (CreERT2; Tsc2fl/fl Tfebfl/fl) normalized body weight and eliminated the cystic renal disease. In conclusion, TFEB hyperactivation is the missing link between TSC and BHD, two diseases with overlapping symptoms in their lung and kidney manifestations, identifying TFEB as a therapeutic target for both.

References: Alesi N, Akl EW, Khabibullin D, Liu H-J, Nidhiry AS, Garner ER, Filippakis H, Lam HC, Shi W, Viswanathan SR. TSC2 regulates lysosome biogenesis via a non-canonical RAGC and TFEB-dependent mechanism. Nature Communications. 2021;12(1):1-12. Napolitano G, Di Malta C, Esposito A, de Araujo MEG, Pece S, Bertalot G, Matarese M, Benedetti V, Zampelli A, Stasyk T, Siciliano D, Venuta A, Cesana M, Vilardo C, Nusco E, Monfregola J, Calcagni A, Di Fiore PP, Huber LA, Ballabio A. A substrate-specific mTORC1 pathway underlies Birt-Hogg-Dube syndrome. Nature. 2020;585(7826):597-602. Epub 20200701. doi: 10.1038/s41586-020-2444-0. PubMed PMID: 32612235; PMCID: PMC7610377.

Presenting Author: Daniel R. Crooks, National Institute of Health, USA

Abstract Title: Proteomic analysis of the pyruvate dehydrogenase complex in metabolomics extracts obtained from Birt-Hogg-Dubé syndrome renal tumors.

Authors and Affiliations: Daniel R. Crooks1,2, Ye Yang1,2, Ronald J. Holewinski3, Christopher J. Ricketts1, Laura S. Schmidt1,4, Cathy D. Vocke1, Thorkell Andresson3, W. Marston Linehan1,2 1Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 2Clinical Cancer Metabolism Facility, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 3Protein Characterization Laboratory, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc, Frederick, MD, USA 4Basic Science Program, Frederick National Laboratory for Cancer Research, Frederick, MD, USA

Abstract text

Background Germline mutations in the FLCN gene cause Birt-Hogg-Dubé syndrome (BHD), an autosomal dominant inherited syndrome in which patients are at risk of developing dermal fibrofolliculomas, pulmonary cysts, and multifocal renal tumors (1). While multiple signaling pathways including AKT-mTOR, AMPK, and TFE3/TFEB transcriptional activation have been implicated in the pathogenesis of BHD tumors (2), few studies have investigated the activity of central metabolite pathways in FLCN-deficient human renal tumors and tumor cells. Methods We utilized NMR- and mass spectrometry-based metabolomics approaches to investigate and characterize central metabolic pathways in the patient-derived FLCN-deficient renal tumor cell line (UOK257) and in renal tumors obtained from BHD patients undergoing surgery at the NIH Clinical Center. We developed a method to perform global proteomics and phospho-proteomics on protein residues obtained during the extraction of cell and tissue samples for polar and non-polar metabolomics analyses. Protein residues were obtained using a three-phase extraction strategy (3), resulting in dried protein residues which were then subjected to trypsinization, TMT derivatization, chromatographic separation with Orbitrap-based detection, database search and post-processing analysis. Results and Conclusions Metabolomics analyses provided evidence for enhanced glucose oxidation and pyruvate dehydrogenase (PDH) activity in BHD tumor tissues relative to renal cortex tissues. Global proteomics analysis of tissue protein residues revealed a significant increase in PDHA1 protein levels in BHD tumors as compared to renal cortex samples (log2 fold change = 0.84, p.

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. and Linehan, W.M. (2005) High frequency of somatic frameshift BHD gene mutations in Birt-Hogg-Dube-associated renal tumors. J Natl Cancer Inst, 97, 931-935. 2 Linehan, W.M., Schmidt, L.S., Crooks, D.R., Wei, D., Srinivasan, R., Lang, M. and Ricketts, C.J. (2019) The Metabolic Basis of Kidney Cancer. Cancer Discov, 9, 1006-1021. 3 Crooks, D.R., Fan, T.W. and Linehan, W.M. (2019) Metabolic Labeling of Cultured Mammalian Cells for Stable Isotope-Resolved Metabolomics: Practical Aspects of Tissue Culture and Sample Extraction. Methods Mol Biol, 1928, 1-27.

Presenting Author: Sophie Gad, Gustave Roussy Institute (GR), France

Abstract Title: The French BHD database: focus on renal tumors.

Authors and Affiliations: Sophie GAD1, Caroline ABADIE2, Nelly BURNICHON2, Sophie COUVE1, Sophie GIRAUD2, Virginie VERKARRE2, Arnaud MEJEAN2, Jean-Michel CORREAS2, Sophie FERLICOT2, Stéphane RICHARD1,2 1) EPHE-PSL, UMR CNRS 9019 Gustave Roussy Paris-Saclay, Villejuif, France 2) Réseau National de Référence pour Cancers Rares de l’Adulte PREDIR, AP-HP/INCa, Hôpital Bicêtre, Le Kremlin-Bicêtre, France

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The French National Network for Rare Cancers in Adults “PREDIR”, coordinated by Prof. Stéphane RICHARD, is in charge of clinical management of patients with hereditary renal cancers and research on related predisposition genes. PREDIR was created in 2009 and is labelled since 2014 by the French NCI (INCa). PREDIR is also certified as International Clinical Care Center by the BHD Foundation. To date 204 distinct families corresponding to 370 BHD patients are followed by PREDIR. FLCN germline mutation was identified in 197 families (96.6%). Cutaneous fibrofolliculomas were present in 226 patients (61%) and pneumothorax occurred in 173 patients (46.7%), respectively. In this cohort, 74 patients (20%) developed renal tumors. The mean age at diagnosis was 49 years (20-83 years). Patients were affected with a spectrum of histological subtypes, including hybrid oncocytic/chromophobe tumor, chromophobe renal cell carcinoma, oncocytoma, papillary renal cell carcinoma and clear cell renal cell carcinoma. The majority of these patients carries a frameshift mutation in the FLCN gene, however 7 mutations were missense variants. This series is one of the most important BHD cohorts and could be used to answer some clinical and fundamental questions in research, that could lead to a better understanding of the disease, new ways of treating it and better management of patients.

References: . Kluger N, Giraud S, Coupier I, Avril MF, Dereure O, Guillot B, Richard S, Bessis D. Birt-Hogg-Dubé syndrome: clinical and genetic studies of 10 French families. Br J Dermatol. 2010 Mar;162(3):527-37.
. Benusiglio PR, Giraud S, Deveaux S, Méjean A, Correas JM, Joly D, Timsit MO, Ferlicot S, Verkarre V, Abadie C, Chauveau D, Leroux D, Avril MF, Cordier JF, Richard S; French National Cancer Institute Inherited Predisposition to Kidney Cancer Network. Renal cell tumour characteristics in patients with the Birt-Hogg-Dubé cancer susceptibility syndrome: a retrospective, multicentre study. Orphanet J Rare Dis. 2014 Oct 29;9:163.
. Bodard S, Boudhabhay I, Dariane C, Delavaud C, Guinebert S, Joly D, Timsit MO, Mejean A, Verkarre V, Hélénon O, Richard S, Correas JM. Percutaneous Thermal Ablation for Renal Tumors in Patients with Birt-Hogg-Dubé Syndrome. Cancers (Basel). 2022 Oct 11;14(20):4969.

Presenting Author: Dr. Xiaowen Hu, University of Science and Technology of China, China

Abstract Title: Clinical and genetic characteristics of 100 patients with Birt-Hogg-Dubé syndrome in Eastern China.

Authors and Affiliations: Daiju Hu1, Rui Wang1,2, Jinli Liu3,, Xianmeng Chen1, Xianliang Jiang4, Jun Xiao5, Wei Wei6, Jay H. Ryu7, Xiaowen Hu1*, 1Department of Pulmonary and Critical Care Medicine, the First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China.  2WanNan Medical College, Wuhu, China. 3Department of Dermatology, the First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China. 4Department of Thoracic Surgery, the First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China. 5Department of Urology, the First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China. 6Department of Radiology, the First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China. 7Division of Pulmonary and Critical Care Medicine, Mayo Clinic, Rochester, MN.

Abstract Text

Background: Although increasing number of patients with Birt-Hogg-Dubé syndrome (BHD) are being recognized in China [1-4], the clinical and genetic characteristics need to be better defined in large cohorts.

Methods: We retrospectively analyzed the data of 100 consecutive patients with BHD encountered at the First Affiliated Hospital of University of Science and Technology of China from Jan 2017 to June 2023.

Results: All 100 patients (63 women) were of Han race with a mean age of 46.8 years. They came from 65 unrelated families in Eastern China, mostly Anhui Province. The common manifestations were pulmonary cysts (99%), pneumothorax (60%) and skin lesions (77%). Renal cancer was detected in only 5 patients and renal angiomyolipoma in 4 others. Sixty-three patients had family history of pneumothorax. The main presentations leading to diagnosis were pneumothorax (38%), family screening (35%), and lung cysts detected on radiologic imaging (25%). The average delay in diagnosis was 7.6 years, and 4.4 years in patients with only pulmonary cysts. In total, 25 FLCN germline mutations were detected among 86 patients, including 7 novel mutations. In addition to hotspot mutation c.1285delC/dupC (17%), the most frequent mutations were c.1015C>T (16%), c.1579_1580insA (14%) and exons 1–3 deletion (11%) in FLCN. The risk of pneumothorax was higher with exons 1–3 deletion mutation and c.1177-5_1177-3de1CTC compared to those with point mutation c.1285dupC (30%), at 91% [p < 0.05] and 67% [p =0.302], respectively.

Conclusions: Pulmonary cysts and pneumothorax were common features, and skin lesions appeared to be more prevalent than previously reported from Asian countries [1,2,5]. The delay in diagnosis remains a challenge in China. BHD patients in Eastern China presented different genotypic characteristics from other races and its association of pneumothorax phenotype warrant further prospective research [5-7].

References: Hu X, Zhang G, Chen X, et al. Birt-Hogg-Dubé syndrome in Chinese patients: a literature review of 120 families. Orphanet J Rare Dis. 2021;16(1):223.

  1. Liu Y, Xing H, Huang Y, et al. Familial spontaneous pneumothorax: importance of screening for Birt-Hogg-Dube syndrome. Eur J Cardiothorac Surg. 2020;57(1):39–45; 2. Liu K, Xu W, Tian X, et al. Genotypic characteristics of Chinese patients with BHD syndrome and functional analysis of FLCN Orphanet J Rare Dis. 2019;14(1):223; 3. Zhang G, Liu J, Wang Y, et al. Birt-Hogg-Dubé syndrome encountered at rare lung disease clinic in Anhui province, China.Orphanet J Rare Dis. 2022;17(1):203; 4. Furuya M, Yao M, Tanaka R, et al. Genetic, epidemiologic and clinicopathologic studies of Japanese Asian patients with Birt-Hogg-Dube syndrome. Clin Genet. 2016;90(5):403-12; 5. Toro JR, Wei MH, et al. BHD mutations, clinical and molecular genetic investigations of Birt-Hogg-Dube syndrome: a new series of 50 families and a review of published reports. J Med Genet. 2008;45(6):321-31; 6. Sattler EC, Syunyaeva Z, Mansmann U, et al. Genetic Risk Factors for Spontaneous Pneumothorax in Birt-Hogg-Dube Syndrome. Chest. 2020;157(5):1199-206.

Presenting Author: Ryosuke Jikuya, Yokohama City University, Japan

Abstract Title: Analysis of tumor cells of origin and driver mutation timing of BHD-associated renal tumors based on novel algorithms.

Authors and Affiliations: Ryosuke Jikuya1,2, Todd A Johnson2, Kazuhiro Maejima2, Ikuma Kato3, Mitsuko Furuya4, Go Noguchi1, Sachi Kawaura1, Yasuhiro Iribe1, Kota Aomori1, Masaya Baba5, Satoshi Fujii3, Kazuhide Makiyama1, Masahiro Yao1, Brian M Shuch6, Christopher J Ricketts7, Laura S Schmidt7,8, W Marston Linehan7, Hidewaki Nakagawa2 and Hisashi Hasumi1 1Department of Urology, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa 236-0004, Japan. 2Laboratory for Cancer Genomics, RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa 230-0045, Japan. 3Department of Molecular Pathology, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa 236-0004, Japan. 4Pathology Center, GeneticLab Co., Ltd., 28-196, N9, W15, Chuo-ku, Sapporo, 060-0009, Japan. 5Laboratory of Cancer Metabolism, International Research Center for Medical Sciences, Kumamoto University, Kumamoto 860-0811, Japan. 6Institute of Urologic Oncology, UCLA School of Medicine, Los Angeles, CA 90095, USA. 7Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. 8Basic Science Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA. Ryosuke Jikuya1,2 Todd A Johnson2, Kazuhiro Maejima2, Ikuma Kato3, Mitsuko Furuya4, Go Noguchi1, Sachi Kawaura1, Yasuhiro Iribe1, Kota Aomori1, Masaya Baba5, Satoshi Fujii3, Kazuhide Makiyama1, Masahiro Yao1, Brian M Shuch6, Christopher J Ricketts7, Laura S Schmidt7,8, W Marston Linehan7, Hidewaki Nakagawa2 and Hisashi Hasumi1.

Abstract Text

Background: BHD-associated renal tumor, caused by alteration of FLCN gene, develops chromophobe renal cell carcinoma (ChRCC) and hybrid oncocytic/chromophobe tumor (HOCT). Meanwhile, sporadic (non-hereditary) ChRCC does not harbor FLCN alteration. There has been much interest in why these renal tumors with different genetic background show the similar histological types. Methods: To elucidate tumorigenesis of these tumors, we performed whole genome sequencing (WGS) and RNA sequencing (RNA-seq) analyses of 16 BHD-associated renal tumors and 21 sporadic ChRCCs. Also, we integrated the cancer genome atlas (TCGA) data for comparative analyses. Results: Compared to sporadic ChRCC, BHD-associated renal tumors showed the upregulation of oxidative phosphorylation, downregulation of cancer-related gene expression, lower tumor mutation burden, higher mitochondrial DNA copy number and lower mitochondrial variant allele frequency (heteroplasmy). Our previous single-cell transcriptome analysis showed intra-tumor heterogeneity in BHD-associated renal tumors based on mutually exclusive expression of L1CAM, a marker of renal collecting duct principal cells, and FOXI1, a marker of renal collecting duct intercalated cells [1]. Interestingly, sporadic ChRCCs exhibited inter-tumor heterogeneity defined by these markers (L1CAM and FOXI1). WGS analysis revealed no common mutations other than FLCN in BHD-associated renal tumors, as well as no common structural variant, suggesting that FLCN mutations are the major driver mutations in the tumorigenic mechanism of BHD-associated renal tumors. Furthermore, our machine learning-based algorithm identified proximal tubular cells as tumor cells of origin in BHD-associated renal tumors, whereas collecting duct intercalated cells in sporadic ChRCCs, indicating that these renal tumors have different tumor cells of origin. Furthermore, mutation timing analysis revealed that complete FLCN deletion in BHD-associated renal tumors occurred in early third decade of life, approximately 30 years before surgery. Conclusions: BHD-associated renal tumor and sporadic ChRCC are different types of tumors arising from different cells of origin and distinct therapeutical approaches are needed for these tumors.

References: [1] Jikuya R, Murakami K, Nishiyama A, Kato I, Furuya M, Nakabayashi J, Ramilowski JA, Hamanoue H, Maejima K, Fujita M, Mitome T, Ohtake S, Noguchi G, Kawaura S, Odaka H, Kawahara T, Komeya M, Shinoki R, Ueno D, Ito H, Ito Y, Muraoka K, Hayashi N, Kondo K, Nakaigawa N, Hatano K, Baba M, Suda T, Kodama T, Fujii S, Makiyama K, Yao M, Shuch BM, Schmidt LS, Linehan WM, Nakagawa H, Tamura T, Hasumi H. Single-cell transcriptomes underscore genetically distinct tumor characteristics and microenvironment for hereditary kidney cancers iScience. 2022 May 25;25(6):104463.

Presenting Author: Damir Khabibullin, Brigham & Women’s Hospital, USA

Abstract Title: Tfe3 regulates Wnt pathway ligands and receptors in Folliculin-deficient lung mesenchymal stromal cells.

Authors and Affiliations: Damir Khabibullin*, Ling Chu**, Yongfeng Luo**, Hui Chen**, Wei Shi**, Elizabeth P. Henske* Presenting Author: Damir Khabibullin *Brigham and Women’s Hospital, Harvard Medical School, Boston MA **University of Cincinnati, Cincinnati, OH

Abstract Text

Birt-Hogg-Dube (BHD) syndrome, caused by loss-of-function mutations in the Folliculin (FLCN) gene, is characterized by benign skin tumors, kidney cancer and cystic lung disease with lung collapse (pneumothorax) (1). FLCN has been linked to multiple signaling networks including mTOR, AMPK and WNT (1) and many of its functions are mediated by the transcription factors TFEB and TFE3, which we previously linked to WNT signaling (2). We previously demonstrated that inactivation of Flcn in lung mesenchyme using Dermo-1-Cre leads to pulmonary cystic disease resembling human BHD (3). In unpublished data, we developed a novel lung mesenchymal stromal cell (MSC) line derived from a Flcn-flox/flox mouse. Flcn deletion was induced by adenoviral-Cre. FLCN-deficient MSCs grew 60% faster compared to controls. Tfe3 or Tfeb downregulation prevented this increase. RNA-sequencing of the MSCs revealed that expression of Wnt pathway ligands and receptors was significantly increased in Flcn-deficient cells, including Wnt7a (over 1,000-fold increase), Wnt10a (37-fold increase), Wnt9a (2.5-fold increase), and Lgr5 (26-fold increase). Wnt signaling plays a critical role during lung development, regulating mesenchyme/epithelium crosstalk in the alveoli. TFE3 downregulation in FLCN-deficient cells reversed 75-80% of the FLCN-dependent transcriptional changes, including all Wnt ligands and receptors. In contrast, TFEB downregulation reversed only 40% of the FLCN-dependent changes. Interestingly, in this cellular context, some FLCN-dependent transcriptional changes were equally dependent on TFE3 and TFEB (such as Igfbp5 and Serpinb7), with near complete normalization by either TFE3 or TFEB downregulation, while other genes were primarily dependent on TFE3. These data suggest that TFE3 is a critical downstream target of FLCN in lung mesenchyme and may play a role in BHD-associated pulmonary cystic disease via dysregulation of Wnt signaling during lung development.

References: 1. Schmidt LS, Linehan WM. FLCN: The causative gene for Birt-Hogg-Dube syndrome. Gene. 2018;640:28-42. 2. Kennedy JC, Khabibullin D, Hougard T, Nijmeh J, Shi W, Henske EP. Loss of FLCN inhibits canonical WNT signaling via TFE3. Human Molecular Genetics. 2019;28(19):3270-81. 3. Chu L, Luo Y, Chen H, et al. Mesenchymal folliculin is required for alveolar development: implications for cystic lung disease in Birt-Hogg-Dube syndrome. Thorax. 2020;75(6):486-93.

Presenting Author: Dr. Laura Schmidt, National Cancer Institute, USA

Abstract Title: PRDM10 RCC is a Birt-Hogg-Dubé-like syndrome with fibrofolliculomas, lipomas and highly penetrant, aggressive papillary renal tumors.

Authors and Affiliations: Laura S. Schmidt1,2, Cathy D. Vocke2, Christopher J. Ricketts2, Zoë Blake2, Kristin K. Choo2, Deborah Nielsen2, Rabindra Gautam2, Daniel R. Crooks2, Krista L. Reynolds2, Janis L. Krolus3, Meena Bashyal3, Baktiar Karim3, Edward W. Cowen4, Ashkan A. Malayeri5, Maria J. Merino6, Ramaprasad Srinivasan2, Mark W. Ball2, Berton Zbar2, W. Marston Linehan2 1Basic Science Program, Frederick National Laboratory for Cancer Research, Frederick , MD USA; 2Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD USA; 3Molecular Histology Laboratory, Frederick National Laboratory for Cancer Research, Frederick, MD USA; 4Dermatology Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD USA; 5 Radiology and Imaging Sciences, Clinical Research Center, National Institutes of Health, Bethesda, MD USA; 6Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD USA.

Abstract Text

Background: The aim of this study was to characterize the clinical manifestations and genetic basis of a familial cancer syndrome in affected members of a family who presented with lipomas and Birt-Hogg-Dubé(BHD)-like clinical manifestations including fibrofolliculomas, trichodiscomas and kidney cancer, but for whom no pathogenic FLCN variant was identified. Methods: Genomic analysis of blood and renal tumor DNA was performed. Phenotypic manifestations and inheritance pattern, and clinical and surgical management were documented. Renal tumor histology and cutaneous and subcutaneous pathologic features were characterized. RNA expression analysis was performed in renal tumor tissue. Results: Affected individuals were found to be at risk for a highly penetrant, aggressive form of bilateral, multifocal papillary renal cell carcinoma. Whole genome sequencing identified a germline pathogenic variant in the PRDM10 gene (c.2029 T>C, p.Cys677Arg), which cosegregated with disease. PRDM10 loss of heterozygosity (LOH) was identified in kidney tumors. PRDM10 was predicted to abrogate expression of FLCN, a transcriptional target of PRDM10, and this was confirmed in the renal tumors by demonstrated loss of FLCN gene expression and elevated expression of GPNMB, a TFE3/TFEB target, and well-documented downstream biomarker of FLCN loss. In addition, a sporadic papillary renal carcinoma from the TCGA cohort was identified with a somatic PRDM10 mutation in the same codon. Conclusions: We identified a germline PRDM10 pathogenic variant in association with a highly penetrant, aggressive form of familial papillary RCC, lipomas and fibrofolliculomas/ trichodiscomas. PRDM10 LOH and elevated GPNMB expression in renal tumors indicate that PRDM10 alteration leads to reduced FLCN expression, driving TFE3-induced tumor formation. These findings suggest that individuals with BHD-like manifestations and subcutaneous lipomas, but without a germline pathogenic FLCN variant, should be screened for germline PRDM10 variants. Importantly, kidney tumors identified in patients with a pathogenic PRDM10 variant should be managed with surgical resection instead of active surveillance. *Funded in part by FNLCR Contract HHSN261201500003I.

Presenting Author: Dr. Rob Wolthuis, Amsterdam UMC, the Netherlands

Abstract Title: Secretome Analysis of Renal Tubular Epithelial Cells Reveals Candidate Extracellular Biomarkers of FLCN Loss in Birt Hogg Dube Syndrome.

Authors and Affiliations: Klaas de Lint1, Fraser Johnson1,2, Iris Glykofridis1, Jesper Balk1, Rodrigo Leite de Oliveria1, Arjan Houweling1, Rob Wolthuis1* Amsterdam UMC, location VUmc, Vrije Universiteit Amsterdam, 1 Human Genetics, 2 Neuro Oncology, Cancer Center Amsterdam, De Boelelaan 1118, 1081 HV Amsterdam, The Netherlands; Klaas de Lint is the presenting author.

Abstract Text

In Birt–Hogg–Dubé (BHD) syndrome, germline mutations in the Folliculin (FLCN) gene predisposes carriers to an increased risk of renal cancer (1). Loss of heterozygosity, by gene silencing or an inactivating somatic mutation of the wildtype FLCN allele, precedes the development of bilateral and multifocal renal tumors in patients with BHD (1). Here, we used pooled genome-wide CRISPR screens to reveal cellular dependencies and functional pathways in renal epithelial cells. To focus on the identification of vulnerabilities or growth regulatory genes associated with FLCN inactivation, we screened RPTEC-hTERT-FLCN-KO-TP53-KO (C2), an aneuploid tumor model system for BHD-RCC, and its counterpart (C2+FLCN) in which FLCN function was restored by cDNA expression. Guide-level gene scores were combined into a normalized gene-level Z-scores, normZ, from which p-values and a false discovery rate were derived. We identified druggable candidate genes which may be specifically required for the survival of FLCN-deficient cells, offering possible leads for future molecular targeting of BHD tumors. Interestingly we also found genes which, upon inactivation, increased the fitness of FLCN-KO RPTEC cells. Based on pathway analyses and integration with existing transcriptomics and proteomics data (2,3), we are building a framework of key factors underlying the cellular consequences of FLCN inactivation. This framework may provide new handles for clinical applications in BHD.

References: 1. Houweling AC, Gijezen LM, Jonker MA, van Doorn MB, Oldenburg RA, van Spaendonck-Zwarts KY, Leter EM, van Os TA, van Grieken NC, Jaspars EH, de Jong MM, Bongers EM, Johannesma PC, Postmus PE, van Moorselaar RJ, van Waesberghe JH, Starink TM, van Steensel MA, Gille JJ, Menko FH. Renal cancer and pneumothorax risk in Birt-Hogg-Dubé syndrome; an analysis of 115 FLCN mutation carriers from 35 BHD families. Br J Cancer. 2011, 105:1912-9. 2. Glykofridis, I.E., et al., Loss of FLCN-FNIP1/2 induces a non-canonical interferon response in human renal tubular epithelial cells. Elife, 2021, 10: p. e61630. 3. Glykofridis IE, Henneman AA, Balk JA, Goeij-de Haas R, Westland D, Piersma SR, Knol JC, Pham TV, Boekhout M, Zwartkruis FJT, Wolthuis RMF, Jimenez CR. Phosphoproteomic Analysis of FLCN Inactivation Highlights Differential Kinase Pathways and Regulatory TFEB Phosphoserines. Mol Cell Proteomics. 2022, 21:100263.