Summary of the Sixth BHD and First International Upstate Kidney Cancer Symposium

Last week more than 100 BHD researchers, clinicians and patients attended the Sixth BHD Symposium and First International Upstate Kidney Cancer Symposium in Syracuse, New York. The Symposium was expertly organised and run by Professor Gennady Bratslavsky, Dr Mehdi Mollapour and their team at SUNY Upstate Medical University. The packed programme featured presentations from several renowned BHD and renal cancer specialists.

In the opening keynote presentation Dr W. Marston Linehan, Chief of the Urologic Oncology Branch at the NCI, NIH, reviewed the genetic basis of kidney cancer and the importance of understanding the underlying biology for development of treatments. The rest of the first day focused on the ongoing BHD and RCC scientific research.

Dr Laura Schmidt reviewed the mutations and presentation of 226 BHD families that are part of the NCI’s programme, and Dr Jorge Toro provided an update on current and emerging techniques to identify FLCN mutations. Dr Elizabeth Henske and Dr Vera Krymskaya focused on FLCN’s function in the lungs and role in cell adhesion that could be associated with cyst formation. Dr Maria Czyzyk-Krzeska discussed a role for FLCN in autophagy targeting the midbody. Dr Mehdi Mollapour discussed interactions between FLCN, FNIP1/2 and the chaperone protein Hsp-90; and Diana Dunn further discussed Hsp90 and the regulation of its co-chaperones in RCC.

A common focus was the impact of altered metabolism in RCC: Dr Damir Khabibullin discussed the role of FLCN-interacting protein PKP4/p0071 in metabolic regulation; Dr Arnim Pause spoke about the impact of increased glycogen production on tumourigenesis both as an energy source and a mechanism to increase hyperosmotic stress resistance; Dr Yu Jiang linked a role for FLCN as a ciliary protein to regulation of mTOR activity and altered metabolism; and Dr Sunil Sudarshan demonstrated that altered metabolism in RCC could also impact cancer cell epigenomes.

Additional presentations detailing model systems for BHD and RCC came from Dr Angela Pacitto who discussed the yeast FLCN/FNIP1 orthologues Lst7/Lst4 and their structural organisation, and Dr Masaya Baba who described the development of a new Xp11.2 translocation mouse model for RCC.

The day ended with a poster session, sponsored by Faculty of 1000, featuring 14 posters from research groups around the world which were assessed by Dr Sunil Sudarshan. First place was awarded to Adam Price, second place to Dr Damir Khabibullin, and third place to Diana Dunn.

The second day of the symposium focused on the clinical science of BHD and RCC and featured a keynote presentation from Dr Robert Uzzo, from the Fox Chase Cancer Centre, on assessing and managing patients’ risk rather than their cancer.

Initially the presentations focused on BHD:  Dr Mitsuko Furuya presented data on the 115 Japanese families seen by BHD-NET; Dr Paul Johannesma and Dr Irma van de Beek discussing pulmonary and renal pathologies in Netherlands patients; and Dr Nishant Gupta presented data supporting the cost-effectiveness of HRCT screening for BHD in patients presenting with a pneumothorax. There were additional presentations from the VHL Alliance’s Ilene Sussman on the CGIP databank and Dr Jo Robays on the Belgian guidelines for BHD.

Subsequently the presentations were focused on more general clinical aspects of RCC with Dr Brian Shuch, Dr Haifeng Yang and Dr Fang Ming Deng discussing the varied presentation and histology of various forms of non-clear cell RCC and the impact classification can have on treatment and outcome. Dr Adam Metwalli spoke about the different surgical approaches needed in patients with multifocal RCC to minimise impact on function and outcome. Then Professor Gennady Bratslavsky stressed that surgeons should also know when not to cut in metastatic patients, and when to offer targeted treatments.

Ongoing clinical trials were discussed by Dr Jason Muhitch, who spoke about the AGS-003 immunotherapy trial (discussed in this blog post) and the potential of high-dose radiation to bolster anti-tumour immune responses, and Dr Namita Chittoria who discussed trials for papillary RCC caused by MET mutations.

Thank you to all those who participated in the Symposium and contributed to its success. The Symposium also included a patient-focused session which will be covered in a later blog post.

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Live Update: Sixth BHD & First Upstate Kidney Cancer Symposium

We are here live at the Sixth BHD Symposium and First Upstate Kidney Cancer Symposium, at the Upstate Medical University in Syracuse, New York. Upstate Medical University has an esteemed record in medical research and continues to focus on major healthcare challenges. The city of Syracuse also offers a beautiful and friendly backdrop to the Symposium.

The Symposium began on Wednesday evening, when attendees met for a cocktail reception in the Tiffany Ballroom of the Genesse Grande Hotel. The reception gave everyone the chance to meet and catch up before the scientific sessions started on Thursday.

After an introduction by one the co-chairs of the scientific organising committee, Dr Mehdi Mollapour, the Thursday morning session began with a keynote speech from Dr W. Marston Linehan of the National Cancer Institute at the NIH. Dr Linehan gave a comprehensive overview of the Genetic Basis of Kidney Cancer. The second co-chair Professor Gennady Bratslavsky presented Dr Linehan with an engraved crystal obelisk to thank him for opening the conference and to acknowledge his contributions to the field.

The rest of Thursday’s talks were focused on the scientific understanding of BHD and renal cancer. There were too many presentations to cover here so look out for the Symposium summaries that will be published shortly. The day ended with a poster session sponsored by Faculty of 1000, where Adam Price – an undergraduate from Dr Maria Czyzyk-Krzeska’s lab – was awarded the Best Poster prize for their work on “VHL and FLCN kidney tumour suppressors are positive regulators of autophagic program targeting midbodies for lysosomal degradation”. Second prize was awarded to Dr Damir Khabibullin and third prize to Diana Dunn. This was followed by dinner which provided more opportunities for networking and discussions.

There are many more talks to come today, these with a focus on the clinical aspects of BHD and kidney cancer. The second keynote speech from urology surgeon Dr Robert Uzzo of the Fox Chase Cancer Centre, will provide an update on the management strategies for renal cell carcinoma.

After these talks the attendees are being treated with a visit to a local winery and dinner at the Belhurst Castle, Geneva. This stone mansion was built on the shores of the Seneca Lake in 1888 during the Romanesque Rivivial. It has a interesting history having been used as a speakeasy and casino during the prohibition, followed by becoming a restaurant before being converted into a hotel.

To end the conference on Saturday morning there will be a patient-focused session led by genetic counsellors Lindsay Middelton and Bonnie Braddock. Here patients will have the opportunity to discuss their condition with clinical experts. A full report from this session will also be posted in the coming weeks.

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Use of Cavitation Ultrasonic Surgical Aspirators for partial nephrectomies

Partial nephrectomies are technically challenging surgeries but preserve healthy renal tissue and therefore function. To minimise bleeding the major blood vessels are usually clamped and tumour extraction completed under ischemic conditions – the renal tissue is deprived of oxygen and nutrients due to restricted blood flow. Although ischemic conditions for less than 25 minutes have minimal reported impact on renal function (Volpe et al., 2015), more complex tumours result in prolonged ischemia making the preservation of healthy tissue more difficult. In recent years several zero-ischemic techniques, including minimally invasive and robotic approaches (Gill et al., 2011, Simone et al., 2013), have been developed that do not require renal artery clamping.

A recent report from Weibl et al. (2015) prospectively assessed 13 partial nephrectomy patients whose tumour removal was achieved using a Cavitation Ultrasonic Surgical Aspirator (CUSA). The patients showed a range of tumour characteristics of varied severities. Weibl et al. assessed operative time, time required for tumour removal, estimated blood loss and the requirement for transfusion. Additionally measures of renal function – estimated glomerular filtration rate and relevant serum protein levels – were collected preoperatively and postoperatively for comparison. CUSA technology has previously been used for partial nephrectomies, although predominantly in animals (Addonizio et al., 1984), and is regularly used in hepatic and neurological surgeries.

CUSA instruments use ultrasonic vibrations to induce cavitation in cells with high water content, such as neoplastic tissue. The formation and subsequent destruction of small bubbles, induced by changes in pressure, results in shockwaves capable of separating cells from tissue. The vibrations can only penetrate up to 2mm through tissue and will cut smoothly through organ tissue without damaging vascular, duct or nerve tissue. The inclusion of an aspirator and electrocautery element enables simultaneous dissection, tissue fragment removal and control of blood loss from smaller vessels. However, larger vessels still require clamping or suturing during dissection to minimise blood loss and maintain visibility.

All of the 13 patients reported in Weibl et al. underwent partial nephrectomies without the need for renal artery clamping. The median blood loss was only 250mL and only one patient required blood transfusion during surgery. Any bleeding was controlled with sutures, and the cut surface was covered with a fibrin sealant adhesive and sutured as required. The medial surgical time was 175 minutes with 12 minutes for tumour removal. Although four of the patients had post-operative complications these were easily managed and are similar to the complications seen after other partial nephrectomy techniques.

A variety of histologies were identified in the tumours including clear cell, papillary and hybrid renal cell carcinomas (RCC), suggesting this method could be effective in the majority of RCC patients. Clear resection boundaries of 1-4mm of healthy tissue around the tumour were confirmed in 12/13 cases with the remaining tumour having a 2-3mm focal positive boundary. There was no decline in renal function in any of the patients post-surgery or in the subsequent three months.

The ability to remove tumours without further reducing kidney function is of particular importance in patients with inherited forms of renal cancer as they are more likely to develop subsequent tumours in either kidney. There are other surgical techniques being developed which also aim to preserve renal function; large randomised trials are required to determine if a particular method, or methods, is best suited to different patients and tumour types. However, this pilot trial from Weibl et al. suggests that the use of CUSAs is feasible and safe for a range of tumour types and severities, and should be further assessed in additional cases.

  • Addonizio JC, Choudhury MS, Sayegh N, Chopp RT (1984). Cavitron ultrasonic surgical aspirator. Applications in urologic surgery. Urology. May;23(5):417-20. PMID: 6719660.
  • Gill IS, Eisenberg MS, Aron M, Berger A, Ukimura O, Patil MB, Campese V, Thangathurai D, Desai MM (2011). “Zero ischemia” partial nephrectomy: novel laparoscopic and robotic technique. Eur Urol. Jan;59(1):128-34. PMID: 20971550.
  • Simone G, Ferriero M, Papalia R, Costantini M, Guaglianone S, Gallucci M (2013). Zero-ischemia minimally invasive partial nephrectomy. Curr Urol Rep. Oct;14(5):465-70. PMID: 23893291.
  • Volpe A, Blute ML, Ficarra V, Gill IS, Kutikov A, Porpiglia F, Rogers C, Touijer KA, Van Poppel H, Thompson RH (2015). Renal Ischemia and Function After Partial Nephrectomy: A Collaborative Review of the Literature. Eur Urol. Jul;68(1):61-74. PMID: 25703575.
  • Weibl P, Shariat SF, Klatte T (2015). Partial nephrectomy driven by cavitron ultrasonic surgical aspirator under zero ischemia: a pilot study. World J Urol. May 3. [Epub ahead of print] PMID: 25935329.
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 Upcoming BHD and Upstate Kidney Cancer Symposium

On September 24-26th the Sixth BHD and First Upstate Kidney Cancer Symposium will be taking place in Syracuse, New York. Hosted by Dr Medhi Mollapour and Professor Gennady Bratslavsky of the Upstate Medical University, it will focus on scientific and clinical developments in BHD and renal cell cancer.

The Symposium will create networking opportunities for clinicians, researchers and patients, enabling the exchange of ideas and discussion of challenges. This will advance the development of innovative and progressive therapies, and improve the management of patients with BHD and multifocal renal cancer.

This year’s first keynote speaker is Dr W. Marston Linehan, Chief of Urologic Oncology at the National Cancer Institute (NCI) in Bethesda. Dr Linehan has been at the forefront of research into inherited forms of kidney cancer for many years with a focus on molecular genetics and translational studies (Linehan & Ricketts, 2014). The second keynote speak is Dr Robert Uzzo, Department of Surgery Co-Leader at Fox Chase Cancer Centre. Dr Uzzo will provide an update on the current management strategies for RCC.

Other prominent BHD researchers presenting include: Dr Laura Schmidt also from the NCI; Dr Maria Czyzyk-Krzeska from the University of Cincinnati; Dr Elizabeth Henske from Harvard Medical School;  Dr Vera Krymskaya from the University of Pennsylvania; Dr Arnim Pause from McGill University; and Dr Jorge Toro from the Washington DC VA Medical Center. Additionally urologic pathologist Dr Fang Ming Deng and VHL Alliance executive director Ilene Sussman will be presenting, with a selection of shorter presentations chosen from submitted abstracts.

The Symposium sessions will focus on basis science on Thursday 24th followed by a poster session, and on surgical and clinical topics on Friday 25th September. Thursday’s range of topics includes FLCN gene function and mutations, pathogenesis in BHD and RCC with regards to cellular pathways such as metabolism and cellular stress, and BHD model systems. Friday’s then focus on patient identification and diagnosis, the clinicopathology of pulmonary and renal manifestations in BHD, and the current management practices and potential future therapies for BHD and RCC.

The conference will end with a patient focused session on the morning of Saturday 26th September. This will include brief presentations and Q&A sessions with genetic counsellors Lindsay Middelton and Bonnie Braddock, alongside several of the pulmonology, dermatology, oncology and pathology experts. Patients and family members will also have the opportunity to discuss their experiences of BHD and gain advice on managing their symptoms. The patients are also invited to attend the earlier conference and social sessions to network with other participants.

For those unable to attend highlights and summaries will be posted on the blog, twitter and facebook. We look forward to seeing all the participants in Syracuse on Thursday 24th.

  • Linehan WM, Ricketts CJ. Decade in review-kidney cancer: discoveries, therapies and opportunities. Nat Rev Urol. 2014 Nov;11(11):614-6. PMID: 25287783.
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Everolimus for the treatment of lymphangioleiomyomatosis

mTOR is dysregulated in a range of tumour types and can be targeted with mTOR inhibitor treatments such as everolimus and sirolimus. Tuberous sclerosis complex (TSC) and sporadic lymphangioleiomyamatosis (LAM) result from mutations in TSC1 or TSC2 that disrupt mTOR signalling (Carsillo et al., 2000, Glasgow et al., 2010). The associated aberrant cell growth, survival and movement results in the formation of slow growing tumours in various tissues and pulmonary cyst formation with loss of pulmonary function. The pivotal role of mTOR signalling in the pathogenesis of TSC/LAM mean mTOR inhibitors have great potential as treatments.

Previous trials have shown that sirolimus and everolimus are effective treatments for angiomyolipomas, slow-growing kidney tumours, in TSC and LAM patients (Bissler et al., 2008, Bissler et al., 2013). It was noted that the angiomyolipoma patients with LAM receiving sirolimus showed improved pulmonary function (Bissler et al., 2008). This effect was confirmed in the larger MILES phase III trial (McCormack et al., 2011). Now a new report from Goldberg et al., (2015) suggests that everolimus could also be an efficient treatment for LAM pulmonary symptoms.

The cystic degeneration of lung tissue in LAM patients eventually results in chronic respiratory failure and can limit survival to 10-20 years after diagnosis. The decline in pulmonary function can be monitored by assessing the decrease in force vital capacity (FVC) and forced expiratory volume in one second (FEV1). It is also associated with an increase in serum VEGF-D levels.

Goldberg et al. assessed everolimus in 24 female patients, with 20 completing the initial 26-week dose escalation trial (2.5mg, 5mg and 10mg daily) and 17 entering the optional extension period of up to 62 weeks. Dose reduction occurred as required in patients with adverse events. The range of adverse events correlated with those reported in previous everolimus trials with severe events only associated with the higher dosage. The authors state that the chosen dosages were based on previous oncology safety trials, and potentially lower doses of everolimus, with fewer adverse events, would be effective.

Everolimus treatment in these patients resulted in FVC stability and improved FEV1; the increased effect on FEV1 compared to FVC is suggestive of reduced airflow obstruction. The patients also showed reduced levels of serum VEGF-D, however there was no correlation between the reduction in VEGF-D and increased lung function. These results provide the initial evidence for everolimus as an effective LAM treatment comparable to other mTOR inhibitors. Everolimus has a shorter half-life and a greater bioavailability than sirolimus, potentially offering a more effective treatment which can more rapidly clear the body if required.

Recently this blog discussed an ongoing phase II clinical trial of everolimus in BHD patients with renal cell carcinoma (RCC). Dysregulated mTOR signalling has also been indicated in pulmonary BHD pathologies with increased mTOR activity reported in patient cystic lung samples (Furuya et al., 2012, Nishii et al., 2013). Therefore, mTOR inhibitor treatments might be useful in treating multiple BHD symptoms. However, unlike LAM, BHD is not a progressive degenerative pulmonary disease and cyst formation rarely has a significant impact on pulmonary function. It is debatable therefore whether the adverse effects associated with long-term mTOR inhibitor treatment would be acceptable to the majority of BHD patients without significant advances in health. Future research and trials will help to clarify the role of mTOR inhibitor treatments in these rare diseases.

  • Bissler JJ, McCormack FX, Young LR, Elwing JM, Chuck G, Leonard JM, Schmithorst VJ, Laor T, Brody AS, Bean J, Salisbury S, Franz DN (2008). Sirolimus for angiomyolipoma in tuberous sclerosis complex or lymphangioleiomyomatosis. N Engl J Med. Jan 10;358(2):140-51. PMID: 18184959.
  • Bissler JJ, Kingswood JC, Radzikowska E, Zonnenberg BA, Frost M, Belousova E, Sauter M, Nonomura N, Brakemeier S, de Vries PJ, Whittemore VH, Chen D, Sahmoud T, Shah G, Lincy J, Lebwohl D, Budde K (2013). Everolimus for angiomyolipoma associated with tuberous sclerosis complex or sporadic lymphangioleiomyomatosis (EXIST-2): a multicentre, randomised, double-blind, placebo-controlled trial. Lancet. Mar 9;381(9869):817-24. PMID: 23312829.
  • Carsillo T, Astrinidis A, Henske EP (2000). Mutations in the tuberous sclerosis complex gene TSC2 are a cause of sporadic pulmonary lymphangioleiomyomatosis. Proc Natl Acad Sci U S A. May 23;97(11):6085-90. PMID: 10823953.
  • Furuya M, Tanaka R, Koga S, Yatabe Y, Gotoda H, Takagi S, Hsu YH, Fujii T, Okada A, Kuroda N, Moritani S, Mizuno H, Nagashima Y, Nagahama K, Hiroshima K, Yoshino I, Nomura F, Aoki I, Nakatani Y (2012). Pulmonary cysts of Birt-Hogg-Dubé syndrome: a clinicopathologic and immunohistochemical study of 9 families. Am J Surg Pathol. Apr;36(4):589-600. PMID: 22441547.
  • Glasgow CG, Steagall WK, Taveira-Dasilva A, Pacheco-Rodriguez G, Cai X, El-Chemaly S, Moses M, Darling T, Moss J (2010). Lymphangioleiomyomatosis (LAM): molecular insights lead to targeted therapies. Respir Med. Jul;104 Suppl 1:S45-58. PMID: 20630348.
  • Goldberg HJ, Harari S, Cottin V, Rosas IO, Peters E, Biswal S, Cheng Y, Khindri S, Kovarik JM, Ma S, McCormack FX, Henske EP (2015). Everolimus for the treatment of lymphangioleiomyomatosis: a phase II study. Eur Respir J. Sep;46(3):783-94. PMID: 26113676.
  • 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. N Engl J Med. Apr 28;364(17):1595-606. PMID: 21410393.
  • 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. Pathol Int. Jan;63(1):45-55. PMID: 23356225.
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Immunotherapy AGS-003 trial in localised renal cell carcinoma

Although targeted therapies have enhanced the efficiency of renal cell carcinoma (RCC) treatment, individual responses are usually limited with few complete remissions reported. An expanding therapy field in RCC is immunotherapy – small molecule and autologous treatments that can modulate the immune system to kill cancer cells. Although to date immunotherapies have only induced a response in a subpopulation of patients, a greater proportion of these responses are long lasting highlighting their potential.

Argos Therapeutics recently announced a pilot trial of their autologous immunotherapy AGS-003 as a neoadjuvant treatment prior to nephrectomy in localised RCC (NCT02170389). AGS-003 uses the patient’s own dendritic cells, differentiated from monocytes collected by leukopheresis, and loads these cells with antigens derived from the patient’s tumour. This is intended to induce a cytotoxic T-cell response against tumour cells.

Normally exposure to a foreign antigen activates helper CD4+ T-cells inducing upregulation of CD40L. CD40L binds to CD40 on immature dendritic cells inducing dendritic cell maturation and antigen processing for presentation to CD8+ cytotoxic T lymphocytes (CTLs). Dysfunction of helper CD4+ T-cells and dendritic cells is often seen in RCC patients resulting in a reduced immune response.

AGS-003 aims to address this reduced response by simulating the presence of CD4+ T-cells ex-vivo. Synthetic CD40L RNA and patient tumour-specific RNA are co-electroporated into the patient’s dendritic cells; providing the proteins required for CD40 activation and antigen presentation. By loading dendritic cells with total tumour RNA multiple different tumour antigens are presented to the immune system reducing the risk of clonal escape.

These activated, tumour-antigen loaded dendritic cells are then returned to the patient’s auxiliary lymph node basin via intradermal injection. There tumour antigens are presented to T-cells inducing the production of tumour-specific CD8+ CTLs. The ligation of CD40 in dendritic cells also results in interleukin-12 (IL-12) production – an essential cytokine required to promote the production of a memory T-cell response thereby increasing the likelihood of a long-term response.

AGS-003, in combination with and comparison to the TKI sunitinib, is currently being trialled in metastatic RCC patients (NCT01582672). Sunitinib has been reported to reduce RCC patient tumour-induced immunosuppression, reducing the levels of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) (Finke et al., 2008, Ko et al., 2009). Therefore it is deemed a suitable choice for combination treatments.

The preceding combinatorial phase II trial (NCT00678119) reported a 62% response rate (n=21) and significant increases in median survival (30.2 months) with 24% of patients surviving longer than five years (Amin et al., 2015). In comparison in previous sunitinib trials only 13% of patients survived for longer than 30 months (Motzer et al., 2013). In addition the adverse effects reported were similar to those in patients receiving only sunitinib and could be resolved with standard management.

The efficiency of AGS-003 treatment to induce a memory T-cell response can be measured by increased production of functional CD8+ CTLs. Comparison between baseline levels and levels after five doses of AGS-003 showed increased production in 71% of patients. The increase in CTL production, compared to baseline, was correlated with overall survival (Amin et al., 2015), suggesting that the response seen was the result of AGS-003 treatment.

The new trial will be the first to assess the ability of AGS-003 to interrupt cancer progression before it spreads and is open to all patients with recurrent or stage I/II RCC. If these and future trials continue to show positive results then AGS-003 could provide an effective alternative treatment for sporadic and inherited forms of localised or metastasised RCC.

  • Amin A, Dudek AZ, Logan TF, Lance RS, Holzbeierlein JM, Knox JJ, Master VA, Pal SK, Miller WH Jr, Karsh LI, Tcherepanova IY, DeBenedette MA, Williams WL, Plessinger DC, Nicolette CA, Figlin RA (2015). Survival with AGS-003, an autologous dendritic cell-based immunotherapy, in combination with sunitinib in unfavorable risk patients with advanced renal cell carcinoma (RCC): Phase 2 study results. J Immunother Cancer. Apr 21;3:14. PMID: 25901286.
  • Finke JH, Rini B, Ireland J, Rayman P, Richmond A, Golshayan A, Wood L, Elson P, Garcia J, Dreicer R, Bukowski R (2008). Sunitinib reverses type-1 immune suppression and decreases T-regulatory cells in renal cell carcinoma patients. Clin Cancer Res. Oct 15;14(20):6674-82. PMID: 18927310.
  • Ko JS, Zea AH, Rini BI, Ireland JL, Elson P, Cohen P, Golshayan A, Rayman PA, Wood L, Garcia J, Dreicer R, Bukowski R, Finke JH (2009). Sunitinib mediates reversal of myeloid-derived suppressor cell accumulation in renal cell carcinoma patients. Clin Cancer Res. Mar 15;15(6):2148-57. PMID: 19276286.
  • Motzer RJ, Escudier B, Bukowski R, Rini BI, Hutson TE, Barrios CH, Lin X, Fly K, Matczak E, Gore ME (2013). Prognostic factors for survival in 1059 patients treated with sunitinib for metastatic renal cell carcinoma. Br J Cancer. Jun 25;108(12):2470-7. PMID: 23695024.
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TDP-43 differentially splices FNIP1

Folliculin interaction protein 1 (FNIP1), through interactions with FLCN, plays a role in a range of cellular processes (Baba et al., 2006). Alternative splicing of FNIP1, under the control of MBNL1, was previously reported in late mesenchymal differentiation (Venables et al., 2013). New research from De Conti et al., (2015) has identified FNIP1 as also being differentially spliced by TDP-43 – a protein associated with neurodegeneration in ALS and fronto-temporal dementia (Neumann et al., 2006).

TDP-43, a hnRNP component, helps regulates both DNA expression and mRNA processing, transport and translation for a wide range of genes (Buratti & Baralle, 2012). The loss of TDP-43 has previously been reported to alter expression and splicing of hundreds of genes (Polymenidou et al., 2011). However, as hnRNPs predominantly regulate splicing in a cooperative manner, it is unlikely that all of these genes are directly affected by TDP-43 levels. In fact very few genes have been shown to be directly affected by TDP-43; these include POLDIP3, CFTR, BIM, BCL2 and TARDBP itself.

De Conti et al. used HEK293 cells in a high throughput screen to identify changes in mRNA splicing directly related to TDP-43 depletion. Endogenous TDP-43 was knocked down using siRNA before attempted rescue with a siRNA-resistance wild type TDP-43 or F4L substitution mutant TDP-43 which cannot bind RNA. Using junction arrays it was possible to identify altered mRNA isoform production; genes that showed altered isoform production in the TDP-43 knockdown cells that could be rescued by TDP-43WT but not TDP-43F4L were deemed to be directly affected. In total 145 genes were identified including a large number involved in RNA binding and splicing, and several associated with apoptosis or cell cycle control.

Only the genes with at least a two-fold change in isoform profile, detectable by RT-PCR, were further characterised – of these the novel genes were STAG2, MADD, FNIP1 and BRD8. Altered splicing due to TDP-43 depletion results in the inclusion of STAG2 exon 30b and BRD8 exon 20, and the exclusion of FNIP1 exon 7 amino acids. However, exclusion of MADD exon 31 forms a premature stop codon leading to truncation and nonsense mediated decay. Using a second loss-of-function TDP-43 mutant (12XQ/N), which better mimics ALS pathology by inducing cytoplasmic aggregation of TDP-43 leading to nuclear depletion, resulted in similar alterations in isoform production. These alternations were also seen in neuroblastoma lines potentiality indicating a relevance in neuronal cells and new starting points for novel therapeutic strategies.

The results reported by De Conti et al. also support roles for TDP-43 in other cellular processes and potentially pathologies including cancer. MADD and STAG2 have roles in regulating apoptosis and cellular division respectfully, and have both been implicated in cancers (Kurada et al., 2009, Postal-Vinay et al., 2012).  FNIP1, through its interactions with FLCN and downstream signalling pathways, plays a role in energy metabolism, cell proliferation and apoptosis – processes perturbed in BHD tumours and other pathologies. The loss of FNIP exon 7 would not be expected to directly disrupt binding to FLCN or AMPK, as this is dependent on the C-terminal (Baba et al., 2006), but there are several phosphorylation sites within this domain that would be lost, potentially altering protein stability and interactions. Further research is required to determine what role, if any, TDP-43-depletion and the associated changes are playing in varied pathologies.

It is unknown whether alternative splicing of FNIP1 occurs in all tissues or whether it has any role in BHD pathology. Potentially different FNIP1 isoforms could unable to stably interact with FLCN, therefore perturbing FLCN-regulated cellular pathways leading to tumourigenesis. However, to date there have been no reports regarding TDP-43 or MBNL1 expression in BHD cells. As high throughput screens and genetic sequencing become more common in research it is quite likely that more interactions between BHD-related and novel proteins will be determined. Such discoveries could increase understanding of BHD pathogenesis and help in the development of new treatments.

  • Baba M, Hong SB, Sharma N, Warren MB, Nickerson ML, Iwamatsu A, Esposito D, Gillette WK, Hopkins RF 3rd, Hartley JL, Furihata M, Oishi S, Zhen W, Burke TR Jr, Linehan WM, Schmidt LS, Zbar B (2006). Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling. Proc Natl Acad Sci U S A. Oct 17;103(42):15552-7. PMID: 17028174.
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