Summary of recent kidney cancer clinical trials

Kidney tumours, if detected early enough, can often be removed surgically without the need for further drug treatments. However, if the primary tumour metastasises traditional chemotherapies and radiotherapies become ineffective and patient survival is limited. In recent years there have been great advances in treatments for metastatic renal cell carcinoma (mRCC) with several targeted treatments now available. However, these targeted treatments show variable response rates and efficacy. This blog summarises recent results from clinical trials assessing new treatments.

The standard first-line treatment for mRCC is an anti-angiogenic and anti-proliferative tyrosine kinase inhibitor (TKI) such as sunitinib or sorafenib. In a recent phase II trial sunitinib was used in combination with trebananib – a peptide-Fc fusion protein that acts on additional pathways to further limit angiogenesis. The combination treatment of trebananib and sunitinib showed an increased response rate and longer progression free survival (PFS) (Atkins et al., 2015).However, it also showed increased toxicity compared to sunitinib alone, with a higher percentage of severe adverse events.

In patients where tumours become resistant to the first-line TKI treatment it is common to use an mTOR inhibitor such as everolimus for the second-line treatment. Recently two trials have suggested that novel TKIs Cabozantinib and Lenvatinib could be provide more effective treatments. Patients on cabozantinib – which acts on VEGFR, MET, RET, KIT and AXL signalling – showed a 42% reduction in progression, extended PFS, and a greater rate of tumour reduction (Choueiri et al., 2015). It is also being trialled as a first line treatment and in combination with other treatments. Lenvatinib – which acts on VEGFR, FGFR, RET, KIT and PDGFR signalling – also enhanced PFS and overall survival (OS) in combination with everolimus compared to everolimus treatment alone (Motzer et al., 2015).

An alternative treatment strategy is immunotherapy which enables the body’s own immune system to more efficiently target tumour cells. Nivolumab is a PD-1 inhibitor that restores T-cell immune activity. In comparison to everolimus as a second line treatment, nivolumab was recently reported to increase overall survival and have a greater impact on tumour shrinkage. Patients receiving nivolumab also had a lower rate of severe adverse events (Motzer et al., 2015b).

Finally it was recently announced that Dr W. Marston Linehan is leading a phase II trial of Berg’s drug BPM 31510 at the NIH. BPM 31510 modulates mitochondrial metabolic networks to reverse the Warburg effect characteristic of tumour cells. Preclinical and early trials results show solid tumour reduction and stable disease with no reported severe adverse events.

The development of new and more efficient treatments for kidney cancer is an active field. Ongoing basic research into the biology of tumourigenesis will enable more specific and targeted drugs to be produced. Being able to select patient cohorts that are more likely to respond to a particular treatment would reduce unnecessary and ineffective treatments which can have severe side effects. This relies on the identification of biomarkers which will be discussed in a further blog post.

  • Atkins MB, Gravis G, Drosik K, Demkow T, Tomczak P, Wong SS, Michaelson MD, Choueiri TK, Wu B, Navale L, Warner D, Ravaud A (2015). Trebananib (AMG 386) in Combination With Sunitinib in Patients With Metastatic Renal Cell Cancer: An Open-Label, Multicenter, Phase II Study. J Clin Oncol. 20;33(30):3431-8. PMID: 26304872
  • Choueiri TK, Escudier B, Powles T, Mainwaring PN, Rini BI, Donskov F, Hammers H, Hutson TE, Lee JL, Peltola K, Roth BJ, Bjarnason GA, Géczi L, Keam B, Maroto P, Heng DY, Schmidinger M, Kantoff PW, Borgman-Hagey A, Hessel C, Scheffold C, Schwab GM, Tannir NM, Motzer RJ; METEOR Investigators (2015). Cabozantinib versus Everolimus in Advanced Renal-Cell Carcinoma. N Engl J Med. 5;373(19):1814-23. PMID: 26406150
  • Motzer RJ, Hutson TE, Glen H, Michaelson MD, Molina A, Eisen T, Jassem J, Zolnierek J, Maroto JP, Mellado B, Melichar B, Tomasek J, Kremer A, Kim HJ, Wood K, Dutcus C, Larkin J (2015). Lenvatinib, everolimus, and the combination in patients with metastatic renal cell carcinoma: a randomised, phase 2, open-label, multicentre trial. Lancet Oncol. 16(15):1473-82. PMID: 26482279
  • Motzer RJ, Escudier B, McDermott DF, George S, Hammers HJ, Srinivas S, Tykodi SS, Sosman JA, Procopio G, Plimack ER, Castellano D, Choueiri TK, Gurney H, Donskov F, Bono P, Wagstaff J, Gauler TC, Ueda T, Tomita Y, Schutz FA, Kollmannsberger C, Larkin J, Ravaud A, Simon JS, Xu LA, Waxman IM, Sharma P; CheckMate 025 Investigators (2015b). Nivolumab versus Everolimus in Advanced Renal-Cell Carcinoma. N Engl J Med. 5;373(19):1803-13. PMID: 26406148
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FLCN modifies the cytoplasmic translocation and aggregation of TDP-43

TDP-43 is a DNA/RNA binding protein whose cytoplasmic aggregation is associated with neuronal death in ALS and frontotemporal lobar degeneration (FTLD). TDP-43 has multiple cellular functions and shuttles between the nucleus and cytoplasm. However, in ALS and FTLD nuclear clearance of TDP-43 results in increased cytoplasmic localisation – a precursor to TDP-43 aggregation and stress granule formation. The mechanisms that regulate TDP-43 transport are not well understood but new research from Xia et al. (2015) has uncovered a role for FLCN in its nuclear export and the formation of stress granules.

TDP-43 is ubiquitously expressed and under normal conditions the majority of the protein is located within cell nucleus, only being sequestered to the cytoplasm under stress conditions. When TDP-43 is overexpressed in HEK293 the majority of cells show predominately nuclear localisation with a small percentage of cells showing cytoplasmic or diffuse localisation. However, concurrent overexpression of GFP-FLCN results in increased mislocalisation of TDP-43 to the cytoplasm. Xia et al. found this was due to enhancing FLCN-dependent nuclear export of TDP-43 rather than disruption of nuclear import. siRNA knockdown of FLCN results in reduced TDP-43 transport to the cytoplasm, even under stress conditions.

In the FLCN overexpression cells TDP-43 and GFP-FLCN colocalise in cytoplasmic punctate structures. Co-immunoprecipitation assays determined that FLCN and TDP-43, both wild type and mutant forms, directly interact in an RNA-independent manner. Xia et al. then used deletion mutation assays to map the interaction sites to FLCN residues 202-299 and TDP-43 RNA Recognition Motif 1 (RRM1).

The TDP-43 punctate structures are associated with the lysosomes and colocalise with ubiquitination and autophagy markers. TDP-43 colocalises with the stress granule marker G3BP1 only when GFP-FLCN is present to induce cytoplasmic TDP-43 localisation. In FLCN-depleted cells under stress TDP-43 dissociates from stress granules and then returns to the nucleus after conditions return to normal. The stress granules in these cells were smaller than those in control cells suggesting a role for TDP-43 in regulating the size of stress granules. As FLCN does not colocalise with G3BP1 it is most likely only indirectly impacting stress granule formation by mediating the cytoplasmic accumulation of TDP-43.

This research suggests a role for FLCN in enhancing the translocation of TDP-43 into the cytoplasm ahead of aggregation and stress granule production – although the exact mechanisms are unknown. Whilst interesting, these results are based on in vitro overexpression assays in a single, non-neuronal cell type. To understand the impact of this role for FLCN in neuronal pathology in ALS and FLTD further research is required to assess the interactions of endogenous TDP-43 and FLCN (Warren et al., 2004) in neuronal cell lines and ex vivo human samples. Additional research using kidney, lung and skin cells would be required to determine any pathological impacts in BHD patients. Studying endogenous proteins, using FLCN-specific antibodies, would also avoid the risk of the GFP tag affecting FLCN structure or function.

Increased AMPK signalling in motor neuron cell lines has also been shown to enhance nuclear clearing of TDP-43 (Liu et al., 2015). However, TDP-43 was found not to be a direct substrate of AMPK. As FLCN binds AMPK it is possible that FLCN is mediating the TDP-43 cytoplasmic localisation reported in response to changes in AMPK activity, and that depletion of FLCN could reduce this mislocalisation. TDP-43 was also recently linked to the alternative splicing of FNIP1 (De Conti et al., 2015) but further research is required to determine the impact of these interactions and any variations in isoform production on either ALS, FTLD or BHD pathology.

  • De Conti L, Akinyi MV, Mendoza-Maldonado R, Romano M, Baralle M, Buratti E. TDP-43 affects splicing profiles and isoform production of genes involved in the apoptotic and mitotic cellular pathways. Nucleic Acids Res. 2015 Oct 15;43(18):8990-9005. PMID: 26261209.
  • Liu YJ, Ju TC, Chen HM, Jang YS, Lee LM, Lai HL, Tai HC, Fang JM, Lin YL, Tu PH, Chern Y. Activation of AMP-activated protein kinase α1 mediates mislocalization of TDP-43 in amyotrophic lateral sclerosis. Hum Mol Genet. 2015 Feb 1;24(3):787-801. PMID: 25256353.
  • Warren MB, Torres-Cabala CA, Turner ML, Merino MJ, Matrosova VY, Nickerson ML, Ma W, Linehan WM, Zbar B, Schmidt LS. Expression of Birt-Hogg-Dubé gene mRNA in normal and neoplastic human tissues. Mod Pathol. 2004 Aug;17(8):998-1011. PMID: 15143337.
  • Xia Q, Wang G, Wang H, Hu Q, Ying Z. Folliculin, a tumor suppressor associated with Birt-Hogg-Dubé (BHD) syndrome, is a novel modifier of TDP-43 cytoplasmic translocation and aggregation. Hum Mol Genet. 2015 Oct 29. pii: ddv450. [Epub ahead of print] PMID: 26516189.
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In response to amino acids yeast FLCN-FNIP orthologues Lst7-Lst4 stimulate TORC1 activity

In eukaryotic cells TORC1 signalling has a key role in controlling cell growth in response to nutritional status. Folliculin (FLCN) and the FNIP proteins regulate mTORC signalling via interactions with Rag family GTPases (Petit et al., 2013, Tsun et al., 2012). Recently Péli-Gulli et al. (2015) reported that the yeast orthologues of FLCN and FNIP, Lst7 and Lst4, form a heterodimer that acts as a GTPase Activating Protein (GAP) for yeast Rag family GTPase Gtr2 . Lst4-Lst7 is the first GAP identified for Gtr2.

Rag family GTPases are heterodimers that cycle between an active and inactive state. In the active stimulating state, GTP is bound to RagA or RagB in mammals, or Gtr1 in yeast, and GDP is bound to RagC or RagD in mammals, or Gtr2 in yeast. Regulating and maintaining these GTP/GDP associations relies on interplay between distinct Guanine nucleotide Exchange Factors (GEFs) and GAPs.

During amino acid starvation TORC1 activity is vastly reduced, and only upregulated upon readdition of amino acids. Péli-Gulli et al. found that loss of either or both Lst4 and Lst7 similarly reduced TORC1 activity after amino acid readdition indicative of a shared biological role in amino acid stimulation of TORC1. Using mutant, GTP-locked variants of Gtr1 and Gtr2 it was determined that Lst4 and Lst7 specifically act upstream of Gtr2.

Péli-Gulli et al. used co-immunoprecipitation assays to confirm direct binding between Lst4 and Lst7.  They showed that, under normal conditions, the Lst4-Lst7 heterodimer is mostly cytoplasmic, but that during amino acid starvation is rapidly recruited to the vacuolar membrane where it is adjacent to, but not associated with, Gtr2. Following amino acid readdition, Lst-4-LSt7 interacts with Gtr2, TORC1 is activated, and Lst4-Lst7 is released from the membrane (Figure 1). Additional TORC1 activity inhibits Lst4-Lst7 localisation to the vacuolar membrane, creating a negative feedback loop. Lst4-Lst7 is a Gtr2-specific GAP and cannot stimulate Gtr1 GTP hydrolysis.

Yeast GTPase3

Figure 1: In yeast cells Lst7-Lst4 is recruited to the vacuolar membrane during amino acid starvation. Following readdition of amino acids Lst7-Lst4 act as a GAP for the GTPase Gtr2 stimulating hydrolysis of bound GTP to GDP. The Rag GTPases can then stimulate TORC1 activity. The Lst7-Lst4 heterodimer is then released into the cytoplasm.

Interestingly, in mammalian cells FLCN helps to regulate both RagA/B and RagC. Petit et al., (2013) reported that FLCN-FNIP1 complexes preferentially bind to GDP-loaded RagA/B, acting as a GEF to stimulate mTORC1 signalling. In contrast Tsun et al., (2012) reported that FLCN-FNIP2 complexes acts as a GAP for RagC to induce mTOR signalling (Figure 2).

Mammal GTPase3

Figure 2: In mammalian cells FLCN-FNIP2 is recruited to the vacuolar membrane during amino acid starvation. Following readdition of amino acids FLCN-FNIP2 act as a GAP for the GTPase RagC stimulating hydrolysis of bound GTP to GDP. FLCN-FNIP1 are a GEF for the GTPase RagA or RagB facilitating the release GDP and binding of GTP. The Rag GTPases can then stimulate mTORC1 activity.

The distinction between FNIP1 and FNIP2 in these complexes could explain the different functions with specific Rag family GTPases. In addition different FLCN domains might be associated with different functions: the C-terminal of FLCN contains a DENN domain (Nookala et al., 2012), a domain typically found in GEFs, whilst Tsun et al. showed that it was the N-terminal domains of FLCN that were required for GAP activity. As the yeast FLCN orthologue Lst7 does not contain the DENN domain, and Lst4 is the only identified FNIP orthologue, it may be only the role for FLCN-FNIP as a RagC/Gtr2 GAP that is conserved.

More research is required to determine how these roles for FLCN and FNIP in regulating amino acid induced TORC1 signalling are related to BHD pathology. Further understanding of the impact of FLCN loss on this regulation and the effects on cell growth could identify new research avenues and therapeutic targets.

  • Nookala RK, Langemeyer L, Pacitto A, Ochoa-Montaño B, Donaldson JC, Blaszczyk BK, Chirgadze DY, Barr FA, Bazan JF, Blundell TL. Crystal structure of folliculin reveals a hidDENN function in genetically inherited renal cancer. Open Biol. 2012 Aug;2(8):120071. PMID: 22977732.
  • Péli-Gulli MP, Sardu A, Panchaud N, Raucci S, De Virgilio C. Amino Acids Stimulate TORC1 through Lst4-Lst7, a GTPase-Activating Protein Complex for the Rag Family GTPase Gtr2. Cell Rep. 2015 Oct 6;13(1):1-7. PMID: 26387955.
  • Petit CS, Roczniak-Ferguson A, Ferguson SM. Recruitment of folliculin to lysosomes supports the amino acid-dependent activation of Rag GTPases. J Cell Biol. 2013 Sep 30;202(7):1107-22. PMID: 24081491.
  • Tsun ZY, Bar-Peled L, Chantranupong L, Zoncu R, Wang T, Kim C, Spooner E, Sabatini DM. The folliculin tumor suppressor is a GAP for the RagC/D GTPases that signal amino acid levels to mTORC1. Mol Cell. 2013 Nov 21;52(4):495-505. PMID: 24095279.
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Pleural covering as an alternative treatment for recurrent pneumothorax

Most BHD patients develop pulmonary cysts and although only 30-35% will suffer a pneumothorax the recurrence rate is very high (Toro et al., 2008). The standard treatment for recurrent pneumothorax is pleurodesis, sometimes accompanied with pleurectomy, which attaches the lung surface to the chest wall thereby reducing the risk of further air leaks. An alternative treatment pioneered by several independent groups of researchers and clinicians in Japan is pleural covering which reinforces the surface of the lung without attachment to the chest wall.

Every year in Japan over 12,000 patients with cystic lung diseases undergo surgery – mostly related to recurrent or intractable pneumothoraces.  An average post-operative recurrence rate of 4-20% highlighted the need for alternative treatments.  Pleurodesis is effective and widely used, but permanently attaching the lungs to the chest wall can reduce respiratory function and make subsequent thoracic surgery more difficult, so alternative non-adhesive treatments were of interest to clinicians and patients (Kurihara et al., 2010).

The pleural covering technique uses a bio-absorbable mesh – either regenerative oxidized cellulose (ROC) mesh or polyglycolic acid (PGA) felt – placed over the surface of the lung and attached using fibrin glue (Ueda et al., 2009, Kurihara et al., 2010). The mesh is absorbed into the pleura reinforcing the remaining lung tissue and unruptured cysts, and sealing air leaks. ROC membranes are less likely to adhere to the chest wall and are associated with fewer post-operative recurrences, compared to PGA felt (Uramoto & Tanaka, 2014).

The covering procedure was originally only used to reinforce excision and staple lines following bulbectomies, and found to reduce bullae regrowth at the staple lines and post-operative recurrence (Sakamoto et al., 2004). More extensive lung covering in LAM patients, to reinforce existing cysts, was found to prevent recurrent pneumothoraces without adhesion to the chest wall or reduction in lung function (Kurihara et al., 2008, Noda et al., 2010).

Data presented by Professor Kurihara at the Fifth BHD Symposium in 2013 (5th BHD Symposium Abstract 14) highlighted the efficacy of pleural covering in BHD patients – 45/46 patients treated with bilateral pleural covering had no post-operative pneumothoraces (<56-month follow-up). Additionally Okada et al., (2015) and Ebana et al., (2015) published pleural covering case studies detailing the treatment of four patients who have suffered no further pneumothoraces (<32 month follow-up). Removing all pulmonary cysts in BHD patients would significantly reduce lung function. Therefore instead the ruptured and thin-walled bulging cysts can be removed before reinforcing both the excision sites and remaining unruptured cysts (Okada et al., 2015).

Pleural covering has also been successful in a range of other cystic lung diseases including bronchiolitis obliterans, pulmonary eosinophilic granulomas (Noda et al., 2011), and AAT-deficiency (Kusu et al., 2012). Although currently not a common practice outside Japan, as more long-term case studies are reported and the efficacy of the treatment becomes clearer pleural covering could become a viable alternative to pleurodesis worldwide for patients suffering from recurrent pneumothorax. In particular the lack of adhesion to the chest wall would be advantageous in patients who are likely to require subsequent surgeries to the lungs, heart or oesophagus.

  • Ebana H, Otsuji M, Mizobuchi T, Kurihara M, Takahashi K, Seyama K (2015). Pleural Covering Application for Recurrent Pneumothorax in a Patient with Birt-Hogg-Dubé Syndrome. Ann Thorac Cardiovasc Surg. Sep 11. PMID: 26370712.
  • Kurihara M, Seyama K, Kumasaka T (2009). Preventing LAM Patients from Recurrent Pneumothorax – An Innovative Surgical Method without Adhesion: Total Pleural Covering Technique (TPC). ATS International Conference 2009 Abstract.
  • Kurihara M, Kataoka H, Ishikawa A, Endo R (2010). Latest treatments for spontaneous pneumothorax. Gen Thorac Cardiovasc Surg. Mar;58(3):113-9. PMID: 20349299.
  • Kusu T, Nakagiri T, Minami M, Shintani Y, Kadota Y, Inoue M, Sawabata N, Okumura M (2012). Null allele alpha-1 antitrypsin deficiency: case report of the total pleural covering technique for disease-associated pneumothorax. Gen Thorac Cardiovasc Surg. Jul;60(7):452-5. PMID: 22544422.
  • Noda M, Okada Y, Maeda S, Sado T, Sakurada A, Hoshikawa Y, Endo C, Kondo T (2010). An experience with the modified total pleural covering technique in a patient with bilateral intractable pneumothorax secondary to lymphangioleiomyomatosis. Ann Thorac Cardiovasc Surg. Dec;16(6):439-41. PMID: 21263428.
  • Noda M, Okada Y, Maeda S, Sado T, Sakurada A, Hoshikawa Y, Endo C, Kondo T (2011). A total pleural covering technique in patients with intractable bilateral secondary spontaneous pneumothorax: Report of five cases. Surg Today. Oct;41(10):1414-7. PMID: 21922367.
  • Okada A, Hirono T, Watanabe T, Hasegawa G, Tanaka R, Furuya M (2015). Partial pleural covering for intractable pneumothorax in patients with Birt-Hogg-Dubé Syndrome. Clin Respir J. Jun 15. PMID: 26073198.
  • Sakamoto K, Takei H, Nishii T, Maehara T, Omori T, Tajiri M, Imada T, Takanashi Y (2004). Staple line coverage with absorbable mesh after thoracoscopic bullectomy for spontaneous pneumothorax. Surg Endosc. Mar;18(3):478-81. PMID: 14752657.
  • Toro JR, Wei MH, Glenn GM, Weinreich M, Toure O, Vocke C, Turner M, Choyke P, Merino MJ, Pinto PA, Steinberg SM, Schmidt LS, Linehan WM (2008). BHD mutations, clinical and molecular genetic investigations of Birt-Hogg-Dubé syndrome: a new series of 50 families and a review of published reports. J Med Genet. 208 Jun;45(6):321-31. Review. PMID: 18234728.
  • Ueda S, Isogami K, Kobayashi S (2009). [Uncomplicated covering technique for preventing the recurrence in the thoracoscopic surgery for pneumothorax]. Kyobu Geka. 2009 May;62(5):381-4. Japanese. PMID: 19425378.
  • Uramoto H, Tanaka F (2014). What is an appropriate material to use with a covering technique to prevent the recurrence of spontaneous pneumothorax? J Cardiothorac Surg. Apr 29;9:74. PMID: 24775221.
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Energy, Superglue and Dominoes: A summary of the Sixth BHD Symposium Patient and Family session

On Saturday 26th September the Sixth BHD and First International Upstate Kidney Cancer Symposium ended with a patient and family session. The session was chaired by Lindsay Middelton, a genetic counsellor from the NCI, and featured presentations and Q&A sessions with several clinical experts.

Dr Jorge Toro, a dermatologist at the Washington DC VA Medical Centre, discussed the different types of skin growths reported by BHD patients and recommended that patients get an annual full body skin examination. As fibrofolliculomas can have significant psychological impact, doctors should investigate treatments with patients; although all current treatments are only temporarily effective.

Professor Elizabeth Henske, a medical oncologist at the Brigham and Woman’s Hospital, summarised current understanding of BHD with “energy, superglue and dominoes”: Folliculin (FLCN) has a role in cellular energy sensing and correcting energy disruptions is a focus of RCC targeted treatments; cells without FLCN become “too stuck together” potentially contributing to lung cyst formation as greater forces would be required to expand the lungs; and as FLCN has roles in multiple pathways its loss has a domino effect on other proteins. Greater understanding of BHD basic science is required to identify new therapeutic targets.

Professor Gennady Bratslavsky, Chair of Urology at SUNY Upstate Medical University, spoke about a surgeon’s role in RCC treatment and the importance of avoiding unnecessary surgeries. As the 3cm rule is effective in BHD patients, and the development of additional tumours is a lifelong risk, operating to remove small tumours could result in patients requiring additional surgeries later. Patients should not be afraid to ask for a second opinion on their case.

Dr Nishant Gupta, a pulmonologist from the University of Cincinnati, described the characteristic lower lung location and oblong shape of BHD lung cysts that can be detected by CT imaging. Pleurodesis fuses the lung surface to the chest wall, reducing the risk of recurrent pneumothorax, but sometimes partially fail over time with subsequent surgeries potentially required. BHD cysts are not degenerative and few new cysts develop over time, therefore regular CT scans are not required.

Dr Laura Schmidt, a researcher at the NCI, discussed current BHD animal models and explained how they increase understanding of pathology. She noted however that these models do not truly represent the human disease and results should be verified in human samples provided by patients. This was followed by reassurance from Dr Steve Landas, a pathologist at SUNY Upstate Medical University, that the urgency for new diagnostic tools and treatments is understood by researchers.

Dr Ilene Sussman, director of the VHL Alliance, described the Cancer in our Genes International Patient (CGIP) databank which could increase understanding of BHD natural history. CGIP is a confidential site where patients can volunteer information on a range of health and lifestyle topics. Anonymised data can then be used by approved researchers to identify new trends and disease associations, potentially answering questions on the impacts of diet and exercise.

Lindsey Middelton concluded the session with a presentation about Clinical Trials and the potential of all patients to contribute to the development of new treatments. Poll results show that prospective participants are frequently concerned about receiving a less effective or placebo treatment, the potential side effects and long-term health risks. However, patient safety is paramount and participants receive very high quality care. In addition, patients in clinical trials can be the first to benefit from new treatments. To ensure they are making an informed choice participants should be told about the potential benefits and risks of the treatment, but also the other options available. It is important that those who do participate follow the treatment plan and maintain contact with the clinical team to report any problems or side effects.

A summary of the scientific and clinical sessions can be found here and thank you again to all those involved in the Symposium and who contributed to its success.

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A role for FLCN in modulating resistance to hyperosmotic stress

Water efflux in response to hyperosmotic stress causes cells to shrink, protein and DNA damage, cell cycle arrest and death. Cells must therefore adapt to changes in external osmolality; one conserved mechanism is to rapidly increase intracellular osmolytes to maintain osmotic homeostasis. New research from Possik et al., (2015) has identified a role for FLCN-1, mediated by AMPK activity, in the maintenance of glycogen stores required for rapid production of the osmolyte glycerol in C.elegans.

AMPK, a key regulator of cellular energy homeostasis, is negatively regulated by FLCN. It was previously reported that flcn-1 loss in C. elegans (flcn-1(ok975) mutants) resulted in AMPK-dependent, increases in glycolysis and autophagy enhancing resistance to metabolic stresses (Possik et al., 2014). To determine if flcn-1 loss also confers resistance to hyperosmotic stress Possik et al. (2015) assessed survival on high concentration NaCl plates. The flcn-1(ok975) worms showed increased survival, reduced shrinkage and a faster recovery from paralysis in comparison to wild type worms. Re-expression of flcn-1 reversed the phenotype confirming its role in increased hyperosmotic stress resistance.

In C. elegans hyperosmotic stress triggers increased glycerol-3-phosphate dehydrogenase-1 (gpdh-1) transcription and production of glycerol from glycogen (Lamitina et al., 2004). Electron microscope imaging of flcn-1(ok975) worms identified a significant increase in the size and number of glycogen deposits in comparison to wild type worms, which were depleted after NaCl treatment (Possik et al., 2015). Glycogen synthesis and degradation are mediated by glycogen synthase (gsy-1) and glycogen phosphorylase (pygl-1) respectively. Inhibition of either enzyme strongly reduces survival following NaCl treatment supporting key roles for glycogen accumulation and breakdown in hyperosmotic stress resistance.

As repression of the C.elegans AMPK-α subunits aak-1 and aak-2 abolished the increased survival, enhanced hyperosmotic stress resistance in flcn-1(ok975) worms must be AMPK-dependent. Loss of aak-1 and aak-2 also reduced glycogen accumulation in both wild type flcn-1 and flcn-1(ok975) worms. This suggests that the increased glycogen accumulation seen in flcn-1(ok975) worms is a result of the chronic AMPK activation due to FLCN-1 loss, but also that AMPK signalling plays a role in normal maintenance of glycogen stores.

Degradation of glycogen produces glucose-6-phosphate, a metabolite that can be converted to glycerol by gpdh-1 or gpdh-2. The flcn-1(ok975) worms showed a two-fold increase in gpdh-1 expression under non-stressed conditions and a higher basal level of glycerol. Additionally a marked increase in both gpdh-1 and gpdh-2 expression was reported after two hours treatment with NaCl. Simultaneous inhibition of gpdh-1 and gpdh-2 was required to strongly reduce the increase in survival in flcn-1(ok975) worms. These results support a key role for glycogen store metabolism by gpdh enzymes in resistance to hyperosmotic stress.

FLCN loss is associated with the development of renal tumours in BHD patients and model systems. Analysis of Flcn-null mouse kidneys and patient tumour samples showed increased glycogen accumulation in comparison to healthy tissue. Glycogen biosynthesis and degradation gene expression levels in human non-BHD renal cell carcinoma samples were also upregulated and expression of 46% of these genes was negatively correlated with FLCN expression.

The breakdown of glycogen stores can also produce the metabolites required for glycolysis, a process that protects cells from multiple metabolic stresses. Therefore increased glycogen stores could play dual roles in tumourigenesis as a source of energy and oncolytes: cells able to survive hyperosmotic or metabolic stresses can accumulate more DNA damage thereby increasing the risk of neoplastic transformation. Greater understanding of how tumour cells resist normal cell death programmes can help in the development of new targeted treatments.

  • Lamitina ST, Morrison R, Moeckel GW, Strange K (2004). Adaptation of the nematode Caenorhabditis elegans to extreme osmotic stress. Am J Physiol Cell Physiol. Apr;286(4):C785-91. PMID: 14644776.
  • Possik E, Jalali Z, Nouët Y, Yan M, Gingras MC, Schmeisser K, Panaite L, Dupuy F, Kharitidi D, Chotard L, Jones RG, Hall DH, Pause A (2014). Folliculin regulates ampk-dependent autophagy and metabolic stress survival. PLoS Genet. Apr 24;10(4):e1004273. PMID: 24763318.
  • Possik E, Ajisebutu A, Manteghi S, Gingras MC, Vijayaraghavan T, Flamand M, Coull B, Schmeisser K, Duchaine T, van Steensel M, Hall DH, & Pause A (2015). FLCN and AMPK Confer Resistance to Hyperosmotic Stress via Remodeling of Glycogen Stores. PLoS genetics, 11 (10) PMID: 26439621.
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Flcn-deficient renal cyst cells can be tumourigenic

Kidney-specific Flcn knockout in mice results in the development of large polycystic kidneys and uremia causing renal failure and death within three weeks (Chen et al., 2008). As this short time frame is insufficient for substantial solid tumour growth Wu et al., (2015) extracted renal cyst cells to assess their tumourigenic potential and response to mTOR inhibitors.

Microtumour and cystic hyperplasia cells from 20-day-old Flcn-knockout mouse kidneys were cultured in vitro for at least 35 passages to establish new cell lines; a process that might preferentially select pre-malignant and malignant cells. Genotyping and western blots confirmed that these lines were Flcn-null. Within one month of inoculation into nude mice the cells developed into tumours – sarcomatoid renal cell carcinomas (SRCC) based on histology. SRCCs are highly malignant neoplasms that can evolve from all RCC subtypes but more commonly from chromophobe RCC (Parada et al., 2006) – the most common form in BHD patients.

Disrupted mTOR signaling has been identified as a cause of renal tumourigenesis and has seen in both BHD cell and animal models associated with the loss of FLCN (Baba et al., 2006, Baba et al., 2008, Hasumi et al., 2009Hartman et al., 2009). Wu et al. reported increased mTOR activity, indicated by phosphorylation of S6, in the allografted Flcn-negative tumours. Sirolimus, an mTOR inhibitor, inhibits tumour cell growth in vitro and in vivo by reducing cell proliferation and angiogenesis. The allografted mice were treated with sirolimus to assess its efficacy on the Flcn-null tumours.

Mice with tumours approximately 200mm3 were treated with 7.5mg/kg sirolimus for 21 days and their tumours grew significantly less than those in untreated controls (average 700mm3 vs. 2200mm3). Tumours treated later but with a higher dose of sirolimus -15mg/kg when 450mm3 – also showed slowed growth. To assess the timing effects on sirolimus efficacy mice with tumours only 150mm3 were treated with the lower dosage; the tumours shrank to average of only 117mm3. Treatment groups each contained 15 treated and 15 control mice.

Although these results are encouraging Wu et al. do not provide detailed information on the age of the individual mice when treatment was started. Without this it is difficult to assess the broad efficacy of sirolimus in these tumours as it is possible that variable tumorigenicity could bias responses; for example the smaller tumours, which had the greatest response, could have been inherently slower growing and potentially more susceptible to inhibition. Wu et al. also propose that, based on the dosage response seen, early treatment with a higher dosage would be more effective at reducing and reversing tumour growth but provide no data to support this. As all mice were euthanised at the end of treatment the long term effects of sirolimus treatment on tumour growth could also not be assessed.

Wu et al. also xenografted human FLCN-null UOK-257 cells into nude mice. The resulting tumours showed slowed growth when mice were treated with the higher sirolimus dose. However, the efficacy was lower than in the allograft animals: the treated tumours began to grow again, albeit slower than control tumours, after an average of 12 days. This suggests that they had escaped sirolimus-associated growth inhibition.

The UOK-257 cell line was derived from a human RCC sample (Yang et al., 2008), and like most cell lines is likely to carry uncharacterised mutations in proliferation pathways. Mutations in alternative tumourigenic pathways would enable the cells to escape inhibition of mTOR-associated growth. As such it is debatable whether such cell lines, which have been biasedly selected and accumulate mutations during in vitro culture, can be accurate representations of patient tumours and provide valid data on tumour development and drug responses.

The advanced RCC guidelines list mTOR inhibitors as a first line option for poor risk patients or as a second line option after TKI treatment (Escudier et al., 2014). However, this work and others provide evidence that mTOR inhibitors – potentially in combination with other inhibitors to limit the possibility of escape via alternative pathways – could be a valid treatment for BHD-associated renal tumours. Therefore, given the different genetic background and histology of BHD tumours, it would be advisable to assessment potential treatments in BHD patients to create specific guidelines.

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