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.
  • Buratti E, Baralle FE. TDP-43: gumming up neurons through protein-protein and protein-RNA interactions. Trends Biochem Sci. 2012 Jun;37(6):237-47. PMID: 22534659.
  • De Conti L, Akinyi MV, Mendoza-Maldonado R, Romano M, Baralle M, Buratti E (2015). TDP-43 affects splicing profiles and isoform production of genes involved in the apoptotic and mitotic cellular pathways. Nucleic Acids Res. Aug 10. pii:gkv814. [Epub ahead of print] PMID: 26261209.
  • Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, Chou TT, Bruce J, Schuck T, Grossman M, Clark CM, McCluskey LF, Miller BL, Masliah E, Mackenzie IR, Feldman H, Feiden W, Kretzschmar HA, Trojanowski JQ, Lee VM (2006). Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science. Oct 6;314 (5796):130-3. PMID: 17023659.
  • Kurada BR, Li LC, Mulherkar N, Subramanian M, Prasad KV, Prabhakar BS (2009). MADD, a splice variant of IG20, is indispensable for MAPK activation and protection against apoptosis upon tumor necrosis factor-alpha treatment. J Biol Chem. May 15;284(20):13533-41.PMID: 19289468.
  • Polymenidou M, Lagier-Tourenne C, Hutt KR, Huelga SC, Moran J, Liang TY, Ling SC, Sun E, Wancewicz E, Mazur C, Kordasiewicz H, Sedaghat Y, Donohue JP, Shiue L, Bennett CF, Yeo GW, Cleveland DW (2011). Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43. Nat Neurosci. Apr;14(4):459-68. PMID: 21358643.
  • Postel-Vinay S, Véron AS, Tirode F, Pierron G, Reynaud S, Kovar H, Oberlin O, Lapouble E, Ballet S, Lucchesi C, Kontny U, González-Neira A, Picci P, Alonso J, Patino-Garcia A, de Paillerets BB, Laud K, Dina C, Froguel P, Clavel-Chapelon F, Doz F, Michon J, Chanock SJ, Thomas G, Cox DG, Delattre O (2012). Common variants near TARDBP and EGR2 are associated with susceptibility to Ewing sarcoma. Nat Genet. Feb 12;44(3):323-7. PMID: 22327514.
  • Venables JP, Lapasset L, Gadea G, Fort P, Klinck R, Irimia M, Vignal E, Thibault P, Prinos P, Chabot B, Abou Elela S, Roux P, Lemaitre JM, Tazi J (2013). MBNL1 and RBFOX2 cooperate to establish a splicing programme involved in pluripotent stem cell differentiation. Nat Commun. 4:2480. PMID: 24048253.
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Sharing genomic data to advance research

It is expected that genetic sequencing will advance both specialised and general healthcare leading to more personalised care based on an individual’s genome. It can be used to identify disease-specific mutations and those associated with more complex conditions. However, as discussed previously on this blog, understanding the effect of a single mutation can be difficult without comparison between healthy and disease patient samples. Sharing patient data accumulated by private and academic labs, voluntary databases, healthcare providers and large scale sequencing efforts such as the 100,000 genomes project, can provide researchers with the larger datasets required for accuracy.

Sharing personal data, in an anonymised form, with research partners can only be done with patient consent. When asked about willingness to share data for research the majority (75-90%) of patients with an existing disease would agree (Genetic Alliance UK, 2014, Darquy et al., 2015 (Europe), Oliver et al., 2015 (USA)) compared to only a third of healthy participants (Sanderson et al., 2015 (USA)). This discrepancy is most likely the result of different motivations between patients and families with a disease – who rely on research to advance understanding and treatment options – and those interested in their own general health.

One of the main reasons given by healthy patients for not wanting to share data is the risk that anonymised data could be re-identified with negative connotations for the patients and their families. This is also a common concern for disease patients where potentially, due to a limited patient population, simply having knowledge of the primary disease and local care provider could enable patient identification. Re-identification is an unavoidable possibility and patients must assess the acceptability of such a risk.

Some databases are also shared with commercial partners and in surveys most rare disease patients were happy with this, acknowledging that treatment development requires such collaborations. However, some expressed concerns over whether the results from commercial research would be made publically accessible for other groups to use or would be kept proprietary and used solely for profiteering (Genetic Alliance UK, 2014, Darquy et al., 2015).

Generally patients report that they would want to know what research is being conducted with their data and have access to any results including incidental findings (Genetic Alliance UK, 2014, Darquy et al., 2015). However, disclosure of incidental findings, which could impact on patient health, should be handled by trained professionals who can explain the potential implications to patients – this may be difficult for some research groups to ensure. Some research programmes therefore will not return this information to patients, whilst others, including the 100,000 genomes project, will only return consequential findings that are actionable (Genomics England). Details regarding disclosure of incidental findings should be included in consent terms.

Patients who agree to share data often give “broad consent” for their data to be shared with approved users with valid research questions, rather than individual consent for every project. For research outside this remit additional consent would be required with patients having the option to decline. An upcoming study by Genentech using Parkinson disease patients’ data submitted to genetic testing company 23andMe will require additional patient consent as it requires in-depth sequence analysis of individual records rather than being based on the existing anonymised and aggregated data (Adam & Friedman 2015).

Whilst sharing genetic data is important for progressing research donors should ensure they are personally comfortable with how, when, who-with and why their own personal data will be shared before consent is given. They should also be aware of if and how incidental findings will be reported, and the potential impact of such results.

  • Adam S, Friedman JM (2015). Individual DNA samples and health information sold by 23andMe. Genet M Jun 18. PMID: 26087174.
  • Darquy S, Moutel G, Lapointe AS, D’Audiffret D, Champagnat J, Guerroui S, Vendeville ML, Boespflug-Tanguy O, Duchange N (2015). Patient/family views on data sharing in rare diseases: study in the European LeukoTreat project. Eur J Hum Genet. Jun 17. PMID: 26081642.
  • Genetic Allience UK (2014). What do patients with rare genetic conditions
    think about whole genome sequencing in the NHS? http://www.geneticalliance.org.uk/docs/final-100kgp-summary-report-12-11-14.pdf
  • Oliver JM, Slashinski MJ, Wang T, Kelly PA, Hilsenbeck SG, McGuire AL (2015). Balancing the risks and benefits of genomic data sharing: genome research participants’ perspectives. Public Health Genomics. 15(2):106-14. PMID: 22213783.
  • Sanderson SC, Linderman MD, Suckiel SA, Diaz GA, Zinberg RE, Ferryman K, Wasserstein M, Kasarskis A, Schadt EE (2015). Motivations, concerns and preferences of personal genome sequencing research participants: Baseline findings from the HealthSeq project. Eur J Hum Genet. Jun 3. PMID: 26036856.
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High-throughput screening to identify synthetic lethal compounds in RCC

Synthetic lethal compounds selectively kill cancer cells by targeting tumour cell-essential processes, but leave healthy cells unharmed. Research is ongoing to identify such compounds in a range of cancers (reviewed in McLornan et al., 2014) including renal cell carcinoma (RCC). New research from Wolff et al., (2015) has identified homoharringtonine (HHT) as synthetically lethal for a subset of clear cell RCC (ccRCC) tumours associated with VHL mutations.

The high-throughput screen completed by Wolff et al. used the 786-O VHL-deficient cell line alongside a VHL-reconstituted isogenic control line. The cell lines were differentially labelled with GFP and mCherry fluorochromes respectively, enabling imaging-based assessment of cell survival in mixed cultures. Mixed cultures better recapitulate the in vivo setting of tumour and healthy cells side-by-side and reduce the variation caused by sequential screening thereby increasing sensitivity.

Approximately 12,800 compounds – including all FDA approved and NIH experimental drugs (UT Southwestern High-Throughput Screening Core) – were screened for synthetic lethality and synergistic activity with mTOR inhibitor sirolimus. 139 compounds significantly reduced the ratio of VHL-deficient to VHL- reconstituted cells with 15 showing reproducible results at concentrations below 5μM. None of the compounds were significantly more effective in combination with sirolimus than without.

The plant alkaloid HHT was chosen for further investigation due to existing FDA approval for treatment of chronic myeloid leukaemia (CML) thereby representing a drug repurposing opportunity. CML studies have identified that HHT most likely inhibits protein synthesis and increases turnover of the anti-apoptotic factor MCL-1 resulting in apoptosis.

HHT at 50nM, a clinically relevant concentration, induced cell death in 30-40% of VHL-defective cells – but under 10% of the VHL-reconstituted cells – within 36 hours, with evidence of apoptosis within 12 hours. Wolff et al. report an upregulation of anti-apoptotic factor Bcl-xL in the VHL-reconstituted cells that was absent from the VHL-deficient cells as previously reported (Devarajan et al., 2001) and would reduce protection from pro-apoptotic signals.

To confirm lethality in vivo Wolff et al. grafted six independent ccRCC cell lines into nude mice treated with either HHT, sirolimus or an empty vesicle control.  The tumour graft lines were derived from individual ccRCC patient samples (Sivanand et al., 2012) and confirmed to carry VHL mutations, however detailed characterisation of the lines is unavailable. Two of the six tumour graft lines responded to HTT showing an observable inhibition of tumour growth (40.3-63.7%) with evidence of necrosis on examination. In comparison the majority of tumours responded to treatment with rapamycin, showing similar signs of necrosis, but not to empty vehicle treatment. As predicted from the cellular screen there was no synergistic action between HHT and sirolimus on tumour growth.

These results indicate that HHT could be repurposed for some cases of VHL-mutated ccRCC.  It is unclear why the 786-O cell line, plus only two of the graft cell lines, were susceptible to HHT, when the other four VHL-mutation tumour graft cell lines were not. HHT’s effectiveness in CML, which is not associated with VHL mutations, suggests that additional modifiers are involved.  Identifying biomarkers in these lines would enable selection of the patients most likely to respond. This also highlights the need to assess multiple cancer-specific cell lines in synthetic lethality screens to compensate for the heterogenetic nature of cancer and identify compounds for other subtypes.

As previously described on the blog high-throughput screens have identified synthetic lethal compounds for VHL, HLRCC and BHD RCC lines. BHD tumourigenesis is reliant on a second FLCN mutation (Vocke et al., 2005) making tumour cells genetically distinct from surrounding tissue. Previous studies have identified treatment with mithramycin or paclitaxel, and the inhibition of Slingshot2 as capable of selectively killing FLCN-null cells. These compounds act in a variety of mechanisms increasing the understanding of FLCN function and tumourigenic pathways. Further screens may identify additional, potentially synergistic, compounds including other pre-approved compounds for potential repurposing.

  • Devarajan P, De Leon M, Talasazan F, Schoenfeld AR, Davidowitz EJ, Burk RD. The von Hippel-Lindau gene product inhibits renal cell apoptosis via Bcl-2-dependent pathways. J Biol Chem. 2001 Nov 2;276(44):40599-605. PMID: 11514546.
  • McLornan DP, List A, Mufti GJ. Applying synthetic lethality for the selective targeting of cancer. N Engl J Med. 2014 Oct 30;371(18):1725-35. . Review. PMID: 25354106.
  • Sivanand S, Peña-Llopis S, Zhao H, Kucejova B, Spence P, Pavia-Jimenez A,Yamasaki T, McBride DJ, Gillen J, Wolff NC, Morlock L, Lotan Y, Raj GV, Sagalowsky A, Margulis V, Cadeddu JA, Ross MT, Bentley DR, Kabbani W, Xie XJ, Kapur P, Williams NS, Brugarolas J (2012). A validated tumorgraft model reveals activity of dovitinib against renal cell carcinoma. Sci Transl Med. Jun 6;4(137):137ra75. PMID: 22674553.
  • Vocke CD, Yang Y, Pavlovich CP, Schmidt LS, Nickerson ML, Torres-Cabala CA, Merino MJ, Walther MM, Zbar B, Linehan WM. High frequency of somatic frameshift BHD gene mutations in Birt-Hogg-Dubé-associated renal tumors. J Natl Cancer Inst. 2005 Jun 15;97(12):931-5. PMID: 15956655.
  • Wolff NC, Pavía-Jiménez A, Tcheuyap VT, Alexander S, Vishwanath M, Christie A, Xie XJ, Williams NS, Kapur P, Posner B, McKay RM, Brugarolas J. High-throughput simultaneous screen and counterscreen identifies homoharringtonine as synthetic lethal with von Hippel-Lindau loss in renal cell carcinoma. Oncotarget. 2015 Jul 10;6(19):16951-62. PMID: 26219258.
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Everolimus: a new treatment for BHD renal cancer?

Last week the US National Cancer Institute announced a phase II clinical trial to test everolimus, a derivative of rapamycin, in BHD patients with renal cell carcinoma (RCC). The trial is also open to sporadic chromophobe RCC (chRCC) patients. Approximately 85% of BHD-RCC is either chRCC or a chromophobe-oncocytoma hybrid (Pavlovich et al., 2002), but there are no effective treatments available for this RCC subtype. Instead BHD patients undergo partial nephrectomies to excise tumours – while not often impacting greatly on renal function, repetitive surgeries can increase morbidity risks. It is hoped that cancer drugs, such as everolimus, can offer a valid alternative treatment.

Rapamycin, originally an immunosuppressant, is appealing as a cancer treatment due to its anti-proliferative properties – a result of mTOR signalling inhibition. Everolimus, and temsirolimus, were forms of rapamycin derived to have improved hydrophilicity (enabling oral and intravenous use), improved pharmacokinetics, and reduced immunosuppressive and toxic effects. They, like rapamycin, bind FKBP2 to inhibit mTORC1 signalling; a pathway found to be upregulated in a wide range of cancers (Moschetta et al., 2014).

The choice to trial everolimus in BHD patients is based on research that has found increased mTOR signalling in patient RCC and lung cyst samples, BHD cell lines and BHD-mouse kidney tumours (Baba et al., 2008, Hasumi et al., 2009, Nishii et al., 2013). In addition, preclinical studies in mouse models have found that treatment with rapamycin can reduce kidney cyst and tumour growth, and extend life span (Baba et al., 2008 , Chen et al., 2015). There have also been several case reports of BHD patients responding well to everolimus as part of their treatment programme (Nakamura et al., 2013, Benusiglio et al 2014) providing further support for the concept of the trial.

However, the relationship between FLCN and mTOR signalling is not fully understood, and may show tissue-specificity, as other groups have reported reduced mTOR signalling in human cell lines and mice renal cysts (Hartman et al., 2009, Bastola et al., 2013). As such everolimus may not be an effective treatment for all, or even any, BHD pathologies.

Everolimus is already approved as a second line treatment for metastatic RCC, some breast and pancreatic cancers, and subependymal giant cell astrocytoma (SEGA) in TSC patients. There are currently several hundred ongoing clinical trials assessing everolimus in a range of cancers and neurological disorders. In addition it is being trialled in LAM patients to assess impact on pulmonary pathologies (Goldberg et al., 2015) – sirolimus has already been found to halt the progression of lung cyst formation (McCormack et al., 2011) and is an approved treatment for angiomyolipomas in LAM and TSC patients. Topical sirolimus can also be used to treat facial angiofibromas in TSC patients (DeKlotz et al., 2011) but a recent trial assessing its use as a fibrofolliculoma treatment produced inconclusive results (Gijezen et al., 2014) – further discussion of this trial can be found here.

FLCN loss perturbs several signalling pathways, so the optimal treatment for BHD-RCC might be a combination of inhibitors. There are ongoing clinical trials accessing the safety and efficacy of combinatorial or sequential treatment of an mTOR inhibitor and a tyrosine kinase inhibitor (TKI), such as pazopanib or sunitinib, in a range of cancers including metastatic RCC. Further research will also increase our understanding of the biological changes responsible for tumour development in BHD and could help in the development of further targeted treatment options.


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Analysing mutational heterogeneity to identify true cancer-associated genes

A recent blog post discussed the need to assess the pathogenicity of genetic variants to determine which mutations are truly causative and which are only background. This is highly important in the search for new cancer drugs as rapidly dividing tissues are more prone to accruing mutations. Large cancer genomic studies are identifying increasing numbers of apparently “significantly-mutated genes” across all major cancer types. However, these genes often include highly unlikely candidates. Analysis of 178 squamous lung cell carcinomas identified 450 significantly-mutated genes; almost a quarter of which were olfactory receptors (TCGA Research Network, 2012). It is necessary to be able to eliminate these false-positives and retain focus on true cancer-genes.

Significantly-mutated genes are identified as harbouring more mutations than expected given the average background mutation rate for the cancer type (Kan et al., 2010). Recent research from Lawrence et al., (2013) has determined significant background mutation frequency heterogeneity across cancer types and the genome as contributing to misidentification of “significantly-mutated” cancer-genes. To account for this heterogeneity they have developed MutSigCV – analytic software which incorporates more sample-specific mutation rates – to help identify which mutated genes are truly associated with cancer.

Lawrence et al. analysed 3,083 tumour-normal pairs across 27 tumour types, including clear cell and papillary Renal Cell Carcinoma (RCC), using whole-exome or whole-genome sequences. They identified 373,909 non-silent coding mutations over all, with an average of 4/Mb and median of 1.5/Mb per sample.

Comparison of individual patient mutation rates within and between cancer types identified variation over 1000-fold. Some variation is based on tumour origin tissue: melanoma and lung cancer samples, from tissues typically exposed to high levels of UV and smoke-based carcinogens respectively, exceeded 100 mutation/Mb compared to paediatric cancers, with less exposure to carcinogens, at around 0.1/Mb. However, there were also magnitudes of difference in variation between patients with the same cancer that could be due to inherited mutations, rather than acquired mutations, driving tumour development.

Lawrence et al. also found regional heterogeneity, up to 5-fold difference, in mutation rates across the genome. There was a strong correlation between high mutation and both low expression rate and late DNA replication. This correlation has been previously reported in germline cells potentially associated with low transcription-coupled repair levels (Fousteri & Mullenders, 2008) and a reduction in available nucleotides later in replication (Stamatoyannopoulous et al., 2009). An increased background mutation rate can explain a large number of the spurious squamous lung cell carcinoma cancer-genes identified as they are both low expression and late replicating genes.

Reanalysis of the original squamous lung cell carcinoma samples using MutSigCV software identified only 11 genes as significantly mutated (TCGA Research Network, 2012). These included genes previous reported to be associated with cancers and one novel gene – HLA-A – suggesting a role for immune evasion in tumourigenesis, an avenue that requires further follow up.

Lawrence et al. acknowledge that there are still other forms of heterogeneity that should be investigated including the co-occurrence of mutations in close proximity and heterogeneity across cancer cells within a tumour. The latter could be important in studies of diseases such as BHD where RCCs of multiple histologies develop and a high proportion of tumours are hybrids (Benusiglio et al., 2014). Although the main tumour driving force in BHD is the loss of FLCN, the development of different tumour types suggests additional mutations resulting in varied histologies. Identifying these mutations could help identify new treatment targets, but to do so it is necessary to separate true cancer-genes from background mutations.

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