This week we would like to introduce you to the work of Professor Vera Krymskaya, Associate Professor of Medicine at the University of Pennsylvania Perelman School of Medicine. Professor Krymskaya’s primary research interest is how signalling pathways cause disease when perturbed, with a focus on the pulmonary diseases Lymphangioleiomyomatosis (LAM) and Birt-Hogg-Dubé (BHD) Syndrome.

Like BHD, LAM is a cystic lung disease caused by an over-proliferation of smooth muscle tissue in the the lungs, mainly affecting women of child-bearing age and individuals with tuberous sclerosis complex (TSC). Professor Krymskaya’s group found that deletion of TSC2 led to the smooth muscle over-proliferation seen in LAM by dysregulation of mTOR signalling. This study also showed that in cell culture the mTOR inhibitor, rapamycin, reduced mTOR signalling and slowed down the growth of TSC2-null cells. This study was the first that suggested that rapamycin could be an effective treatment for LAM, and formed the scientific basis of the MILES trial, which showed rapamycin is an effective treatment for pulmonary LAM (Goncharova et al., 2002; McCormack et al., 2011). This study used cells taken directly from LAM patients’ lungs and grown in culture; Professor Krymskaya’s lab remains the only group in the world to successfully perform this procedure using cells from LAM patients.

While rapamycin halts the growth of lung cysts it does not reverse the lung damage seen in LAM, and has to be taken continuously to stop the disease progressing (McCormack et al., 2011). However, long-term use of rapamycin has been shown to have some severe side-effects, such as hypercholesterolemia and mucositis (McCormack et al., 2011). More recently, Professor Krymskaya’s team have shown that combined therapy of rapamycin and simvastatin in a mouse model of LAM not only stopped the progression of the disease, but that simvastatin seemed to partially reverse the lung damage seen in these animals (Goncharova et al., 2012). Simvastatin is widely used to reduce cholesterol and is not known to have any common adverse side-effects with long-term use. Therefore, if simvastatin proved effective in the treatment of LAM in clinical trials, it could be made available to all LAM patients very quickly.

Hoping to reproduce her success in LAM, Professor Krymskaya’s work on BHD aims to elucidate how FLCN mutations cause cyst formation in the lungs, with the expectation that finding this mechanistic link will suggest potential therapies. Indeed, Professor Krymskaya’s team co-authored a recent study reporting that FLCN interacts with PKP4 to regulate RhoA signalling (Medvetz et al., 2012; Nahorski et al., 2012), suggesting that progress in this area is already being made.

To find out more about Professor Krymskaya and her work at the University of Pennsylvania, please watch our video interview (with its accompanying transcript and audio-only files). Video interviews are also available with Professor Frank McCormack, who also works on LAM and was instrumental in the set up of the MILES trial; and Dr Doug Medvetz and Professor Elizabeth Henske who led the FLCN and RhoA signalling study.

• Goncharova EA, Goncharov DA, Eszterhas A, Hunter DS, Glassberg MK, Yeung RS, Walker CL, Noonan D, Kwiatkowski DJ, Chou MM, Panettieri RA Jr, & Krymskaya VP (2002). Tuberin regulates p70 S6 kinase activation and ribosomal protein S6 phosphorylation. A role for the TSC2 tumor suppressor gene in pulmonary lymphangioleiomyomatosis (LAM). The Journal of biological chemistry, 277 (34), 30958-67 PMID: 12045200
• Goncharova EA, Goncharov DA, Fehrenbach M, Khavin I, Ducka B, Hino O, Colby TV, Merrilees MJ, Haczku A, Albelda SM, & Krymskaya VP (2012). Prevention of alveolar destruction and airspace enlargement in a mouse model of pulmonary lymphangioleiomyomatosis (LAM). Science translational medicine, 4 (154) PMID: 23035046
• McCormack FX, Inoue Y, Moss J, Singer LG, Strange C, Nakata K, Barker AF, Chapman JT, Brantly ML, Stocks JM, Brown KK, Lynch JP 3rd, Goldberg HJ, Young LR, Kinder BW, Downey GP, Sullivan EJ, Colby TV, McKay RT, Cohen MM, Korbee L, Taveira-DaSilva AM, Lee HS, Krischer JP, Trapnell BC; National Institutes of Health Rare Lung Diseases Consortium & MILES Trial Group (2011). Efficacy and safety of sirolimus in lymphangioleiomyomatosis. The New England journal of medicine, 364 (17), 1595-505 PMID: 21410393
• Medvetz DA, Khabibullin D, Hariharan V, Ongusaha PP, Goncharova EA, Schlechter T, Darling TN, Hofmann I, Krymskaya VP, Liao JK, Huang H & Henske EP (2012). Folliculin, the Product of the Birt-Hogg-Dube Tumor Suppressor Gene, Interacts with the Adherens Junction Protein p0071 to Regulate Cell-Cell Adhesion. PloS one, 7 (11) PMID: 23139756
• Nahorski MS, Seabra L, Straatman-Iwanowska A, Wingenfeld A, Reiman A, Lu X, Klomp JA, Teh BT, Hatzfeld M, Gissen P, & Maher ER (2012). Folliculin interacts with p0071 (plakophilin-4) and deficiency is associated with disordered RhoA signalling, epithelial polarization and cytokinesis. Human molecular genetics, 21 (24), 5268-79 PMID: 22965878

www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.

Scurvy was a debilitating ailment that commonly affected sailors in the 18^th Century. In 1747, James Lind conducted one of the first ever clinical trials, by giving sailors with scurvy different dietary supplements and documenting the effects on their health. In commemoration of James Lind’s work, International Clinical Trials Day is celebrated on the 20^th May each year.

A clinical trial aims to answer a medical question and requires patient participation to do so. The question being asked is usually whether a new treatment – most often a drug – is better than the current gold standard of treatment, although other types of trials, such as observational trials and patient preference trials do take place. If you would like more information about how clinical trials are conducted and specific information about trials currently open to BHD patients, please visit our new Clinical Trials page for patients.

It has been estimated to cost $1 billion to get a drug to market (DiMasi et al., 2003). Although this figure has been called into question by Sir Andrew Witty of GSK, the vast majority of clinical trials are still run by large pharmaceutical companies. Rivalry within the industry has caused a culture of secrecy to prevail and data from clinical trials – particularly negative results – are often not published. This makes it extremely difficult for doctors to make fully informed decisions about which drugs to prescribe their patients. Additionally, the amount of regulatory process and paperwork required to initiate new trials is often a major stumbling block for researchers who are new to the process.

In recent years, calls to make clinical trial data available – such as the Alltrials petition spearheaded by Dr Ben Goldacre, author of Bad Pharma – have been heard by pharmaceutical companies, and a number of companies, including GSK and Roche, have drawn up policies on how they will improve the transparency of clinical data. Furthermore, the Health Research Authority (HRA) – a UK-wide steering committee founded in 2011 – aims to streamline the regulatory process involved with starting a new trial, while maintaining rigour and safety. The HRA has also recently announced plans to collaborate with pharmaceutical companies to improve the transparency of clinical trial data. The increase of freely available data, coupled with reducing the barriers to launching new trials, will hopefully lead to more life-saving treatments becoming available to patients more quickly.

Access to clinical trials is often difficult for patients due to strict eligibility criteria. Additionally, many patient advocate groups feel that they have not been included in the design of clinical trials, both of which may contribute to the fact that nearly a third of clinical trials close early because they cannot enrol enough patients to make the data statistically significant (data presented by Jaime Richardson of Cedars-Sinai Hospital at the Kidney Cancer Association conference, May 2013). If this happens in Phase III, the result is that an effective drug is not approved for general use purely because the trial was badly designed. This was described by Ms Richardson, as “heart-breaking and wasteful.”

In the UK, the Health and Social Care Act of 2012, aims to make access to clinical trials an integral part of patient care pathways within the NHS, which will hopefully increase patient participation in trials. Patient education is also an invaluable part of this process and the need for patient advocate organisations to provide information about clinical trials to their patients is paramount. A number of information resources for both patients and researchers are now available online, and several of these can be found towards the bottom of our new Clinical Trials page.

Recognising the importance of educating patients about clinical trials has led to the development of a number of initiatives to support patients wanting to learn more. For example, Cedars-Sinai Hospital in Los Angeles recently created the role of Clinical Trials Recruitment Navigator, which is currently held by Jaime Richardson. Jaime provides patients with information about appropriate trials, and acts as a point of contact and support for those participating in trials. Meanwhile, in the UK the National Institute for Health Research (NIHR) has launched the “Ok to ask” campaign, which promotes the message that it’s ok for patients to ask their doctor about clinical trials.

However, the above plans and initiatives will be in vain if the public perception of clinical trials is not improved. Currently, many patients are understandably wary of participating in a clinical trial as they are worried about receiving a placebo treatment, or that they will be treated as a guinea-pig. Additionally, clinical trials are seen by many as a last resort; only to be considered once all other lines of treatment have failed. In reality, clinical trials are sometimes the only way to access the best drugs and because patients are monitored so closely during the trial, they often receive a better standard of care. Placebo treatments in clinical trials are increasingly rare, and are never used in cancer clinical trials, due to the ethical implications of giving someone who is ill a fake treatment. Contrary to the opinion that clinical trials are a last resort, instead they should be considered as another treatment option upon diagnosis: prior treatment can often make a patient ineligible for a clinical trial, whereas if the clinical trial drug proves ineffective, it is always possible to leave the trial and start traditional treatments before the disease has progressed.

The only trials open to BHD patients at present are observational (details can be found at the bottom of our Clinical Trials page). As BHD is a rare disease, there is currently relatively little data to accurately determine the chances of an individual with a FLCN mutation developing skin lesions, pulmonary cysts, or kidney cancer. As previously discussed, determining this epidemiological data accurately would be a great help in being able to advise newly diagnosed patients of their likely disease progression. There are also several other symptoms, such as parotid lesions and colon cancer, which may be associated with BHD but cannot be conclusively proven at this stage. Observational trials may allow clinicians to conclusively determine whether these symptoms are a risk in BHD syndrome.

Looking to the future, as the profile of rare diseases is raised within the research and pharmaceutical communities, more clinical trials testing potential cures for rare diseases are likely to be launched. The role of patient advocate groups will prove fundamental to this process, as in the case of the Multicenter International LAM Efficacy of Sirolimus (MILES) trial (McCormack et al., 2011) for which The LAM Foundation’s involvement was pivotal in recruiting patients.

Of course, participation in a trial must always be the patient’s decision, but the choice to not participate must be due to “informed refusal” rather than to a lack of access or information. The reasons for participation – or not – are complicated, visceral and utterly personal, and rightly so. But it is always worth bearing in mind that today’s drugs were yesterday’s clinical trials; were it not for James Lind’s efforts in 1747, we wouldn’t know that citrus fruits are a cheap and effective cure for scurvy.

• DiMasi JA, Hansen RW, & Grabowski HG (2003). The price of innovation: new estimates of drug development costs. Journal of health economics, 22 (2), 151-85 PMID: 12606142
• McCormack FX, Inoue Y, Moss J, Singer LG, Strange C, Nakata K, Barker AF, Chapman JT, Brantly ML, Stocks JM, Brown KK, Lynch JP 3rd, Goldberg HJ, Young LR, Kinder BW, Downey GP, Sullivan EJ, Colby TV, McKay RT, Cohen MM, Korbee L, Taveira-DaSilva AM, Lee HS, Krischer JP, Trapnell BC, National Institutes of Health Rare Lung Diseases Consortium, & MILES Trial Group (2011). Efficacy and safety of sirolimus in lymphangioleiomyomatosis. The New England journal of medicine, 364 (17), 1595-606 PMID: 21410393

www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.

The signalling diagram has been updated to include the following recent research papers:

• FNIP2 causes MNU-induced apoptosis (Sano et al., 2013)
• FLCN inhibits MMP9 (Pimenta et al., 2012)
• FLCN inhibits HIF-1a, mTORC1 and mTORC2 (Nishii et al., 2013)
• FLCN and PTEN compound heterozygosity causes oncogenesis (Pradella et al., 2013)
• FLCN and SSH2 are synthetically lethal (Lu et al., 2013)
• FLCN inhibits Cyclin D1 (Kawai et al., 2013)
• FLCN inhibits HIF1a (Gharbi et al., 2013)

Since the Myrovlytis Trust was founded in 2007, there has been growing momentum in the field of BHD research, which is nicely demonstrated in how the signalling diagram has evolved in that time.

The first version of the signalling diagram was posted in March 2011, to coincide with the launch of BHDSyndrome.org. This diagram showed how FLCN slots neatly in to a signalling pathway that includes other genes known to be mutated in kidney cancer, but at this stage there was relatively little known about the functions of FLCN itself.

Version 1 – March 2011

The second version of the signalling diagram was posted a year later in April 2012, adding six new studies to the pathway. In that year, more had been discovered about FLCN’s role in mTOR signalling, and a role for FLCN and its interacting partner FNIP2 in programmed cell death was added to the diagram. The biggest change between versions 1 and 2 of the diagram however, was the increase in information regarding the post-translational modifications of FLCN and its interacting partners. And thus, the first pop-up box was added to display these post-transciptional modifications seperately.

Version 2 – April 2012

The third version of the signalling diagram was published just nine months later in January 2013, incorporating data from a further 15 studies. In those months, a number of hitherto unknown roles for FLCN were reported and the structure of the C-terminus of FLCN was published in August 2012. Additionally, the discovery of several new interacting partners of FLCN necessitated the addition of a second pop-up box to show the structure of FLCN and its interactome. The number of additional proteins and pathways on the diagram made it very busy, and thus all proteins not directly interacting with FLCN were faded out. This allowed the focus of the diagram to be on FLCN and its function, while still retaining the contextual information of FLCN’s position within the “kidney cancer pathway” that formed the core of the original signalling diagram.

Version 3 – January 2013

This month’s update, version 4, incorporates data from the seven new studies listed above. When the diagram was first published in March 2011 it referenced 14 studies, whilst the current version references 42 and soon it will not be possible to fit new information into the diagram in its current form, meaning that a redesign of the diagram will be required. It is greatly encouraging that the amount of BHD research is clearly increasing in pace and volume to make this re-design necessary; increased knowledge about FLCN’s function will  provide insights into how FLCN mutations cause BHD Syndrome, and this will hopefully lead to the development of a cure.

Version 4 – May 2013

• Sano S, Sakagami R, Sekiguchi M, & Hidaka M (2013). Stabilization of MAPO1 by specific binding with folliculin and AMP-activated protein kinase in O⁶-methylguanine-induced apoptosis. Biochemical and biophysical research communications, 430 (2), 810-5 PMID: 23201403
• Pimenta SP, Baldi BG, Nascimento EC, Mauad T, Kairalla RA, & Carvalho CR (2012). Birt-Hogg-Dubé syndrome: metalloproteinase activity and response to doxycycline. Clinics (Sao Paulo, Brazil), 67 (12), 1501-4 PMID: 23295609
• Nishii T, Tanabe M, Tanaka R, Matsuzawa T, Okudela K, Nozawa A, Nakatani Y, & Furuya M (2013). Unique mutation, accelerated mTOR signaling and angiogenesis in the pulmonary cysts of Birt-Hogg-Dubé syndrome. Pathology international, 63 (1), 45-55 PMID: 23356225
• Pradella LM, Lang M, Kurelac I, Mariani E, Guerra F, Zuntini R, Tallini G, Mackay A, Reis-Filho JS, Seri M, Turchetti D, & Gasparre G (2013). Where Birt-Hogg-Dubé meets Cowden Syndrome: mirrored genetic defects in two cases of syndromic oncocytic tumours. European journal of human genetics : EJHG PMID: 23386036
• Lu X, Boora U, Seabra L, Rabai EM, Fenton J, Reiman A, Nagy Z, & Maher ER (2013). Knockdown of Slingshot 2 (SSH2) serine phosphatase induces Caspase3 activation in human carcinoma cell lines with the loss of the Birt-Hogg-Dubé tumour suppressor gene (FLCN). Oncogene PMID: 23416984
• Kawai A, Kobayashi T, & Hino O (2013). Folliculin regulates cyclin D1 expression through cis-acting elements in the 3′ untranslated region of cyclin D1 mRNA. International journal of oncology, 42 (5), 1597-604 PMID: 23525507
• Gharbi H, Fabretti F, Bharill P, Rinschen M, Brinkkötter S, Frommolt P, Burst V, Schermer B, Benzing T, & Müller RU (2013). Loss of the Birt-Hogg-Dubé gene product Folliculin induces longevity in a hypoxia-inducible factor dependent manner. Aging cell PMID: 23566034

As briefly mentioned in last week’s blog, the inaugural conference of the International Rare Disease Research Consortium (IRDiRC) was held in Dublin earlier this month. The conference brought together researchers, clinicians, policy makers and patient organisations from across the globe to share recent successes in the field of rare disease research and to discuss how the field should develop in order to make therapies accessible to patients as quickly as possible.

IRDiRC aims to develop 200 new therapies and the means to diagnose most rare diseases by 2020, and has committed to spend €500 million in order to achieve this goal. Indeed, since IRDiRC was founded in 2010, 64 new rare diseases therapies have been developed through IRDiRC projects, meaning that excellent progress is already being made. However, given that there are estimated to be between 6000-8000 rare diseases, even at this rate, it would take hundreds of years to develop therapies for all of these diseases.

To address this problem, the NIH has set up a National Centre for Advancing Translational Sciences (NCATS), which was introduced to the consortium by Dr Christopher Austin. Because the field of rare diseases is so large and varied, a “one size fits all” approach will not be effective in developing therapies for every disease. Rather than focussing on a particular disease, NCATS researches new methods of developing therapies. One such project they are working on in collaboration with the Office for Rare Disease Research (ORDR) is the development of a tissue chip, containing human tissues and “wired up” to mimic organ function. Once developed, this chip will be used for preliminary toxicity tests for new drugs, meaning that the most dangerous drugs will not reach clinical trials and be tested in people.

Another encouraging development reported at the conference was data showing that gene therapies are providing clinical benefit in patients. Dr Katherine High spoke about the use of adeno-associated virus vectors, which can be used to correct gene function in patients with genetic disorders. The first treatment of this type, Glybera, was licensed in Europe in November 2012 for individuals with Lipoprotein Lipase Deficiency (Kastelein et al., 2013). There are currently more than 1000 phase III gene therapy trials listed on clinicaltrials.gov, suggesting that this is likely to prove a viable cure for a number of rare diseases.

A major theme of the conference was the need for collaboration at all levels to effectively tackle such a complicated and far-reaching problem as rare diseases. Firstly, researchers and clinicians must collaborate to share resources and knowledge. To this end, both IRDiRC and NCATS actively seek to facilitate and fund large-scale collaborative projects. Secondly, patient registries need to be set up in order to ensure patients are getting the correct care and treatment, and to identify patients that might be eligible to participate in research or clinical trials. Additionally, patient registries are essential for natural history and epidemiology studies, which have thus far not been possible for the majority of rare diseases. Setting up a useful patient registry will require multicentre collaboration and infrastructure requiring input from patients, clinicians and researchers to ensure it serves the needs of all parties. Thirdly, patients need to collaborate with one another. Lesley Murphy of RARE Voices, Australia, spoke of the need to unify the activities of all rare disease organisations into a single entity, providing a single, clear message and greatly increasing the success of lobbying and advocacy activities.

The take-home message of the conference was one of great hope, as the founders and members of IRDiRC are committed and passionate about effecting change in the care and treatment of patients with rare diseases. It is expected that this will be achieved through increased funding of rare diseases projects and by developing a culture of collaboration. With 64 rare disease therapies having been developed since 2010, it seems that IRDiRC is already changing the lives of patients with rare diseases.

• Kastelein JJ, Ross CJ, & Hayden MR (2013). From Mutation Identification to Therapy: Discovery and Origins of the First Approved Gene Therapy in the Western World. Human gene therapy PMID: 23578007

www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.

The aim of this year’s Rare Disease Day, Rare Disorders Without Borders, was to promote the message that international collaboration between patients, clinicians and researchers is imperative to find cures for rare diseases. Indeed, this has been the feeling of patients and researchers for some time, and this week’s blog discusses some new projects and collaborations that have been developed to find cures and support those with rare diseases.

The Seventh Framework Programme for Research and Technological Development (FP7) is an EU initiative that has funded around 100 collaborative rare disease research projects since 2007. One such project is EUROPLAN, involving clinicians, researchers, health authorities, and patient organisations from 34 countries, to develop care plans for rare diseases. Based on the recommendations made by EUROPLAN, the EU has now called for all member states to draw up and implement national care plans for rare diseases by the end of 2013, ensuring that access to health care and treatment for rare diseases will be standardised throughout all EU member states.

Founded in 2010, the International Rare Disease Research Consortium (IRDiRC) is a network of rare disease researchers and funders. IRDiRC aims to develop 200 new therapies and the means to diagnose most rare diseases by 2020 and has committed to spend €500 million on rare disease research. The consortium had its inaugural conference in Dublin last week, and will be the subject of next week’s blog.

Historically, pharmaceutical companies have been reticent to develop drugs for rare diseases, so-called orphan drugs, as they are unlikely to recoup the cost of developing the drug. The Orphan Drug Act of 1983 – and similar initiatives in Europe and Japan – set out financial and operational incentives to encourage companies to develop orphan drugs (O’Conner, 2013). Currently the research published on orphan drugs is scattered throughout the literature and can be difficult to find. A new journal, Expert Opinion on Orphan Drugs, collates research on orphan drugs into a single place and will provide reviews and editorials from experts in the field. Additionally, two further journals focussed on rare disease research the Journal of Rare Disorders and Rare Diseases, have also been launched this year. Together, these three journals nearly double the number of journals dedicated to rare disease and orphan drug research.

The lack of interest in rare diseases from pharmaceutical companies could be, in part, due to the term “rare”, which could be interpreted to mean “not of broad clinical significance” or “unprofitable”. A recent article by Dr Anil Mehta, says that rare diseases should be considered as extreme versions of common diseases, and should instead be called “sentinel diseases”. Dr Mehta illustrates his point using the example of BHD Syndrome and suggests that by determining how FLCN mutations cause kidney cancer in BHD syndrome, we will inevitably learn how other forms of kidney cancer develop.

This sentiment was echoed several times at the IRDiRC conference and is embodied by the ethos of Findacure, a funder of rare disease research. Findacure believe that fundamental disease mechanisms underlie both common and rare diseases, and will be best elucidated – and ultimately cured – by studying the most extreme form of these diseases, which are usually the rarer forms. Thus, Findacure refer to these diseases as “fundamental diseases” rather than “rare”.

It is also becoming increasingly clear that drugs developed for common ailments can be re-purposed to treat rare diseases (McCormack et al., 2011), meaning that the reverse is likely to be true. Thus, rare disease research will also lead to insights into the causes of and new interventions for common diseases, making it an increasingly attractive area for pharmaceutical research and investment and hopefully speeding up the development of much needed therapies for common and rare diseases alike.

• McCormack FX, Inoue Y, Moss J, Singer LG, Strange C, Nakata K, Barker AF, Chapman JT, Brantly ML, Stocks JM, Brown KK, Lynch JP 3rd, Goldberg HJ, Young LR, Kinder BW, Downey GP, Sullivan EJ, Colby TV, McKay RT, Cohen MM, Korbee L, Taveira-DaSilva AM, Lee HS, Krischer JP, Trapnell BC, National Institutes of Health Rare Lung Diseases Consortium, & MILES Trial Group (2011). Efficacy and safety of sirolimus in lymphangioleiomyomatosis. The New England journal of medicine, 364 (17), 1595-606 PMID: 21410393
• O’Connor, D. (2013). Orphan drug designation – Europe, the USA and Japan Expert Opinion on Orphan Drugs, 1 (4), 255-259 DOI: 10.1517/21678707.2013.769876

www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.

Several signalling pathways – namely the mTOR, HIF and insulin signalling pathways – are known to slow ageing and increase longevity under certain conditions. This is a topic of much research, and was discussed at the recent “Talks about TORCs” meeting mentioned in last week’s blog. A recent study by Gharbi et al. has now shown that FLCN also regulates longevity in the nematode worm, C. elegans.

As shown in our signalling diagram, VHL functions in similar pathways to FLCN and its loss has previously been shown to increase longevity in C. elegans (Mehta et al., 2009; Müller et al., 2009). With this in mind, Gharbi et al. decided to investigate whether the C. elegans homologue of FLCN, which they identified as F22D3.2, also regulates longevity. In two separate experiments, the authors used a deletion mutant and RNAi knock-down to ablate FLCN function in C. elegans. Both types of FLCN mutant lived for 25 days – an increase of 19% compared to wild type nematodes, which usually live for 21 days, demonstrating that the loss of FLCN increases longevity significantly.

Increased HIF signalling and reduced insulin signalling both increase longevity in C. elegans, Drosophila and mice (Leiser and Kaeberlein, 2010; Parrella and Longo, 2010). Thus, Gharbi et al. wanted to determine whether perturbation of either of these pathways was responsible for the increased lifespan observed in FLCN-null worms. RNAi inhibition of hif-1 ablated the increased longevity of FLCN-null worms, whilst RNAi inhibition of daf-2 and daf-16, components of the insulin signalling pathway, increased longevity by up to an additional 15 days. The authors hypothesised that the increased longevity seen was due to increased stress resistance and tested this by exposing worms to high temperatures (35^oC) for up to ten hours. FLCN-depleted worms showed increased survival compared to wildtype worms under these conditions and, as in the longevity assays, the observed thermo-resistance required HIF signalling and not insulin signalling. Together, these data indicate that FLCN regulates longevity via HIF signalling and independently of insulin signalling.

FLCN-null worms in this study also displayed a mild developmental delay. FLCN can control cell cycling, as discussed here, suggesting it may be a dysregulation of this process causing the developmental delay observed in these worms. Indeed, as the lifespan of C. elegans is precisely 21 days under normal conditions, the delayed development and increased lifespan of FLCN-null animals could represent a generalised “slowing down” of cell division, growth and metabolism. Alternatively, as discussed in a previous blog, FLCN- null cells are known to escape apoptosis and thus the extended lifespan observed in these animals may be analogous to FLCN-depleted cells in BHD patients evading cell death and forming cysts and tumours. Additionally, autophagy, which is induced by HIF signalling, also regulates lifespan in C. elegans and has been shown to become dysregulated in polycystic kidney disease in mice and – as previously discussed – to require FLCN interacting protein 1 (FNIP1) in human embryonic kidney cells (Belibi et al., 2011; Schiavi et al., 2013; Zhang et al., 2008). Therefore, increased autophagy could also be responsible for the extended lifespan of FLCN-null nematodes.

Determining the exact mechanism through which FLCN deletion increases longevity in C. elegans is likely to shed light on mechanisms that contribute to tumour formation in BHD patients. Furthermore, lifespan may prove a useful readout to interrogate FLCN’s interactions with other genes, pathways or molecules in C. elegans, making it an informative model in which to investigate BHD.

• Belibi F, Zafar I, Ravichandran K, Segvic AB, Jani A, Ljubanovic DG, Edelstein CL (2011). Hypoxia-inducible factor-1α (HIF-1α) and autophagy in polycystic kidney disease (PKD). American Journal of Physiology – Renal Physiology, 300 (5), 1235-43 PMID: 21270095
• Gharbi H, Fabretti F, Bharill P, Rinschen M, Brinkkötter S, Frommolt P, Burst V, Schermer B, Benzing T, & Müller RU (2013). Loss of the Birt-Hogg-Dubé gene product Folliculin induces longevity in a hypoxia-inducible factor dependent manner. Aging cell PMID: 23566034
• Leiser SF, Kaeberlein M (2010). The hypoxia-inducible factor HIF-1 functions as both a positive and negative modulator of aging. Biological Chemistry, 391 (10), 1131-7 PMID: 20707608
• Mehta R, Steinkraus KA, Sutphin GL, Ramos FJ, Shamieh LS, Huh A, Davis C, Chandler-Brown D, Kaeberlein M (2009). Proteasomal regulation of the hypoxic response modulates aging in C. elegans. Science, 324 (5931), 1196-8 PMID: 19372390
• Müller RU, Fabretti F, Zank S, Burst V, Benzing T, Schermer B (2009). The von Hippel Lindau tumor suppressor limits longevity. Journal of the American Society of Nephrology, 20 (12), 2513-7 PMID: 19797165
• Parrella E, Longo VD (2010). Insulin/IGF-I and related signaling pathways regulate aging in nondividing cells: from yeast to the mammalian brain. The Scientific World Journal, 10, 161-77 PMID: 20098959
• Schiavi A, Torgovnick A, Kell A, Megalou E, Castelein N, Guccini I, Marzocchella L, Gelino S, Hansen M, Malisan F, Condò I, Bei R, Rea SL, Braeckman BP, Tavernarakis N, Testi R, Ventura N (2013). Experimental Gerontology, 48 (2), 191-201 PMID: 23247094
• Zhang H, Bosch-Marce M, Shimoda LA, Tan YS, Baek JH, Wesley JB, Gonzalez FJ, Semenza GL. Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia. Journal of Biological Chemistry, 283 (16), 10892-903 PMID: 18281291

www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.

Last month, the Third Crick Symposium was held in London (UK), with the aim of discussing ways in which basic biological research could progress from “Genetics to molecules to therapies”. In particular, this meeting brought together chemists, biologists and clinicians from what will be the new Francis Crick Institute in London, which is scheduled to open in 2015.

Of note, Professor Charles Swanton (CRUK London Research Institute and UCL Cancer Institute, UK) described four cases of intra-tumour heterogeneity in metastatic renal cell carcinoma (RCC). These findings demonstrated that certain mutations were ubiquitous, shared or unique within specific regions of the metastatic RCC (Gerlinger et al., 2012). For example, using exome sequencing, chromosome aberration analysis and DNA ploidy profiling it was noted that VHL was mutated in all analysed tumour regions within one patient. An activating mutation in mammalian target of rapamycin (mTOR) was also observed in all but one primary tumour region. However, there were distinct SETD2 and KDM5C mutations present within the primary and metastatic regions of the RCC, and these genes have been previously discussed here. This study by Gerlinger et al. highlights that a single biopsy may not represent the mutational load within a tumour, and that certain tumour cell populations may react differently to treatments. Accordingly, therapies which target ubiquitous mutations may prove to be more successful. It would be particularly interesting to see if there is similar intra-tumour heterogeneity within BHD-associated RCCs, as alluded to in this earlier blog post.

The FLCN-associated signalling diagram and the work described above underlines the importance of mTOR complex (mTORC) signalling in the development of RCC. Thus in mid-March, we attended a set of “Talks about TORCs” organised by the Biochemical Society in London. Recent advances in TOR signalling were shared and its role in a variety of processes was introduced. For example, the first talk by Professor Michael Hall (University of Basel, Switzerland) discussed TOR signalling in relation to growth and metabolism. Notably, Professor Hall suggested that mammalian TOR should be renamed mechanistic TOR, as the pathway is not unique to mammals. This was aptly demonstrated by Dr Miguel Navarro (Institute of Parasitology and Biomedicine “López-Neyra”, Spain) with his work on TOR signalling in Trypanosoma brucei (Barquilla et al., 2012). In addition, Professor Thomas Weichhart (Medical University of Vienna, Austria) and Professor Doreen Cantrell (University of Dundee, UK) introduced the role of mTOR signalling in immunity, which is of interest as FLCN/FNIP1 may be associated with B-cell development (as discussed here and here). Professor Linda Partridge (UCL, UK) also introduced the connection between mTOR signalling and ageing. This is especially relevant as recent work has connected FLCN with longevity in C. elegans (Gharbi et al., 2013), which will be discussed in our BHD Research Blog soon. Moreover, Dr Andrew Tee presented his work on ULK1 and mTOR signalling (Dunlop et al., 2011), as well as a poster on BHD. For more information regarding the work of Dr Tee, please look at our lab profile here.

Finally, do visit our Conferences and Events page to keep updated with meetings that are of relevance to BHD syndrome. In particular, the Fifth BHD Symposium and Second HLRCC Symposium will be held in Paris on 28-29^th June 2013, and the abstract and earlybird registration deadline is 15^th April 2013.

• Barquilla A, Saldivia M, Diaz R, Bart JM, Vidal I, Calvo E, Hall MN, Navarro M. Third target of rapamycin complex negatively regulates development of quiescence in Trypanosoma brucei. Proc Natl Acad Sci U S A. 2012 Sep 4;109(36):14399-404. doi: 10.1073/pnas.1210465109.
• Dunlop EA, Hunt DK, Acosta-Jaquez HA, Fingar DC, & Tee AR (2011). ULK1 inhibits mTORC1 signaling, promotes multisite Raptor phosphorylation and hinders substrate binding. Autophagy, 7 (7). PMID: 21460630
• Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E, Martinez P, Matthews N, Stewart A, Tarpey P, Varela I, Phillimore B, Begum S, McDonald NQ, Butler A, Jones D, Raine K, Latimer C, Santos CR, Nohadani M, Eklund AC, Spencer-Dene B, Clark G, Pickering L, Stamp G, Gore M, Szallasi Z, Downward J, Futreal PA, Swanton C. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med. 2012 Mar 8;366(10):883-92. doi: 10.1056/NEJMoa1113205. Erratum in: N Engl J Med. 2012 Sep 6;367(10):976.
• Gharbi H, Fabretti F, Bharill P, Rinschen M, Brinkkötter S, Frommolt P, Burst V, Schermer B, Benzing T, Müller RU. Loss of the Birt-Hogg-Dubé gene product Folliculin induces longevity in a hypoxia-inducible factor dependent manner. Aging Cell. 2013 Apr 9. doi: 10.1111/acel.12081.

www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.