Nonsteroidal sulfamate derivatives as new therapeutic approaches for Neurofibromatosis 2 (NF2)
Neurofibromatosis 1 and 2, although involving two different tumour suppressor genes (neurofibromin and merlin, respectively), are both cancer predisposition syndromes that disproportionately affect cells of neural crest origin. New therapeutic approaches for both NF1 and NF2 are badly needed. In promising previous work we demonstrated that two non-steroidal analogues of 2-methoxy-oestradiol (2ME2), STX3451(2-(3-bromo-4,5-dimethoxybenzyl)-7-methoxy-6-sulfamoyloxy-1,2,3,4-tetrahydroisoquinoline), and STX2895 (7-Ethyl-6-sulfamoyloxy-2-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahydroisoquinoline) reduced tumour cell growth and induced apoptosis in malignant and benign human Neurofibromatosis 1 (NF1) tumour cells. In earlier NF1 mechanism of action studies we found that in addition to their effects on non-classical hormone-sensitive pathways, STX agents acted on the actin- and myosin-cytoskeleton, as well as PI3Kinase and MTOR signaling pathways. Tumour growth in NF2 cells is affected by different inhibitors from those affecting NF1 growth pathways: specifically, NF2 cells are affected by merlin-downstream pathway inhibitors. Because Merlin, the affected tumour suppressor gene in NF2, is also known to be involved in stabilizing membrane-cytoskeletal complexes, as well as in cell proliferation, and apoptosis, we looked for potentially common mechanisms of action in the agents’ effects on NF1 and NF2. We set out to determine whether STX agents could therefore also provide a prospective avenue for treatment of NF2.
STX3451 and STX2895 were tested in dose-dependent studies for their effects on growth parameters of malignant and benign NF2 human tumour cell lines in vitro. The mechanisms of action of STX3451 and STX2895 were also analysed.
Although neither of the agents tested affected cell growth or apoptosis in the NF2 tumour cell lines tested through the same mechanisms by which they affect these parameters in NF1 tumour cell lines, both agents disrupted actin- and myosin-based cytoskeletal structures in NF2 cell lines, with subsequent effects on growth and cell death.
Both STX3451 and STX2895 provide new approaches for inducing cell death and lowering tumour burden in NF2 as well as in NF1, which both have limited treatment options.
KeywordsNeurofibromatosis 2 Nonsteroidal sulfamate derivatives Tumour treatment Cytoskeleton
malignant peripheral nerve sheath tumours
Both Neurofibromatosis 1 and 2 (NF1 and NF2) are disorders characterized by the formation of tumours of the peripheral and central nervous system , primarily affecting cells of neural crest origin . Although other organ systems and cell types are affected in both NF1 and NF2, the cell of origin in most malignancies is the Schwann cell . Both NF disorders arise through autosomal dominant inheritance with loss-of-function mutations in the tumour suppressing functions of the respective tumour suppressor genes: Neurofibromin (NF1) and Merlin (NF2) [3, 4].
Neurofibromatosis type II (NF2) is associated with loss-of-function mutations in the NF2 gene that encodes the multi-functional protein, Merlin (Moesin-Ezrin-Radixin-like protein) , also known as Schwannomin. Merlin is currently an out-group member of the ERM (Ezrin-Radixin-Moesin) protein family because it is the only one in the family to function as a tumour suppressor. Strong evidence suggests that Merlin regulates the assembly of apico-lateral junctional complex . Merlin is also involved in stabilizing membrane-cytoskeletal complexes , in cell proliferation [8, 9, 10], and in apoptosis . Conditional knockouts of Merlin result in the formation of meningiomas . Conditional deletion of Merlin also contributes to hyperplasia of Schwann cells and of neural-crest derived odontoblasts, osteoblasts, and renal tubular cells. It also results in metastases of osteoscarcoma and fibrosarcoma . Loss of Merlin activates several mitogenic pathways including Rac1/Pak [13, 14], Ras/Raf, PI3K/AKT, mTORC1 and Wnt/β-catenin pathways [15, 16]. Merlin also mediates the Hippo pathway and inhibits proliferation, acting in the nucleus to bind E3 ubiquitin ligase CRL4DCAF1 .
NF2 affects one in 25,000–30,000 live births worldwide. A hallmark of the disease is the formation of bilateral vestibular Schwannomas, as well as the formation of multiple meningiomas, extramedullary spinal tumours, and ependymomas . Uncontrolled growth of these tumours can also lead to cataracts, hearing loss, balance issues and paralysis [5, 6, 19]. Although malignant transformations of NF2 tumours are rare, better therapeutics are needed, because numerous tumours can lead to early morbidity and early mortality (age 36) .
Current treatment options for NF2 tumours include surgical resection of either part of or the complete tumour, which is difficult to perform without damaging nerves. Stereotactic radiosurgery is also an option, however the risk of malignant transformation rises several years post-surgery [20, 21]. Alternate treatment options for NF2 tumours include inhibitors of the epidermal growth factor receptor (EGFR) , inhibitors of the vascular endothelial growth factor (VEG-F) [23, 24, 25], inhibitors of mTORC1 , an inhibitor of platelet-derived growth factor (PDGF) , and an inhibitor of histone deacetylase (HDAC) . However, such treatments have resulted in mixed and sometimes limited success in human trials . Current phase II clinical trials explore better treatment options through inhibition of the mTORC1, PDGF-R, VEGF and anti-angiogenic pathways (NCT01419639; NCT00561665; NCT00589784; NCT02104323). To date, no phase III clinical trials for the treatment of NF2-related disorders have been initiated.
Previous studies from our laboratories  demonstrated that sulfamate ester derivatives of a class of nonsteroidal tetrahydroisoquinoline (THIQ)-derived agents, derived by SAR translation from the naturally occurring anticancer metabolite of 17-β estradiol, known as 2-methoxyoestradiol (2ME2) [31, 32] are highly effective at reducing cell viability in hormone-responsive NF1 cell lines. These derivatives, known as STX3451 and STX2895, are capable of inducing apoptosis in cell lines derived from two malignant peripheral nerve sheath tumours (MPNST) at very low concentration .
Here, we show that NF2 status plays a role in the effectiveness of these non-steroidal 2ME2 derivatives on cell viability in vitro. STX2895 and STX3451 treatment on NF2 null (−/−) cell lines induces nuclear fragmentation, microtubular disruption, inhibition of cellular migration/wound healing, and can induce apoptosis in both benign and malignant NF2 tumour-derived cells.
NF2-null malignant meningioma KT21-MG1-Luc5D cells (KT21 cells, derived from a human malignant meningioma cell line KT21-MG1 ) and the luciferase-expressing NF2-deficient benign meningioma cell line, Ben-Men-1-LucB (Ben-Men-1 cells, derived from a human benign meningioma cell line Ben-Men-1 ), have been described previously . CH157MN cells (a human malignant meningioma NF2−/− cell line from a 41-year-old woman ) were generously provided by Dr. Yancey Gillespie at the University of Alabama Birmingham. HEI-193 cells (a benign vestibular Schwannoma cell line from a 56-year-old Neurofibromatosis type 2 (NF2) patient ) were kindly provided by Dr. Xandra Breakefield, Massachusetts General Hospital. The IOMM-Lee (a malignant Meningioma NF2+/+ cell line derived from the brain of 61-year-old man ) was obtained from Dr. Randy Jensen at the University of Utah. BJ (ATCC® CRL-2522™, human foreskin fibroblast) cells were a gift from Dr. Sem Hin Phan, University of Michigan. Most cell lines were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) (Gibco), supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals, Flowery Branch, GA), 2 mM L-glutamine and 100 U/ml penicillin/streptomycin (Gibco), at 37 °C in a humidified 5% CO2 incubator . HEI-193 cells were maintained in DMEM/10% FBS with forskolin (Cayman Chemical, Ann Arbor, MI), recombinant glial growth factor 2 (R&D Systems, Minneapolis, MN), and geneticin (Gibco). CH157MN cells were grown in MEM/F12 medium with 7% FBS.
STX3451 and STX2895 were synthesized according to previously published protocols [31, 45]. Pak inhibitors, Frax1036 and PF3758309 were used as control to be compared with the effects of STX3451 and STX2895 on NF2-deficient cells. Frax1036 was purchased from Afraxis Inc., San Diego, CA . PF3758309 was synthesized according to published protocols .
Cell viability assays
Cells were plated in 96-well Primaria™ plates (BD Falcon,1 × 104/well) determined by a hemocytometer. STX3451 or STX2895 at concentrations of 100 nM, 300 nM, 600 nM, and 1000 nM was dissolved in vehicle (DMSO; Sigma-Aldrich, St Louis, MO, USA) and were added to the wells 2 hours after plating the cells. The final concentration of DMSO was 1%. PF3758309 (PF) and Frax1036 concentrations ranged from 0.03 μM to 20 μM and from 2.5 μM to 25 μM, respectively. The final concentration of DMSO was 2.5%. STX3451, STX2895, PF, or Frax1036 was replenished after 2 days of culture. Cell growth was assessed using CellTiter 96 (Promega, Madison, WI, USA) [30, 47, 48].
40,000–80,000 cells plated on 0.1% porcine gel-coated coverslips were treated with DMSO, STX3451, STX2895, PF3758309, or Frax1036 for various time periods. Immunocytochemistry and counter-staining with DAPI (4′,6-diamidino-2-phenylindole, Molecular Probes, Eugene, OR), were carried out as reported previously . Microtubules were visualized using antibodies for α-tubulin (1:500, Sigma). Apoptosis was assessed by an anti-annexin V antibody (1:500, Abcam ab14196). Fluorescent (Alexa Fluor 488 or 594) anti-rabbit or anti-mouse secondary antibodies (Life Technologies) were used for visualizing the labeled cells. Fluorescence micrographs were taken with an Olympus BX-51 microscope and processed with Adobe Photoshop.
The ability of cells to migrate was assessed by wound-healing assays [49, 50]. 2 × 105 cells were plated in a 10 mm cell culture dish and allowed to grow to confluence. Wounds were made using a ruler to maintain a straight edge and a 200 μl pipette tip applied to the bottom of the dish to remove the cells. The culture plates were then washed twice with PBS. Fresh medium with DMSO, STX3451, or STX2895 was added to the dishes. STX3451 and STX2895 were used at 300 nM for KT21, Ben-Men-1, and HEI-193 cells, 1000 nM for IOMM-Lee and BJ cells. Photographs of the wounds and subsequent cell migration and “wound filling” were taken using a Nikon SMZ1500 dissecting microscope and digital camera (Nikon Digital Sight) at the time points indicated. The dimensions of the “wound” were measured with ImageJ software.
Cell lysates were collected after 24 h treatments with DMSO, STX3451, STX2895, PF, or Frax1036. Western blotting was performed as described in the studies on NF1 . Membranes were incubated with antibodies (all from Cell Signaling Technology) to phosphorylated S6, S6, cyclin D, phosphorylated MEK, MEK, Pak 1/2, phospho-Pak1 (pPak1), phospho-Pak2 (pPak2) and GAPDH. The peroxidase-coupled secondary antibody (1:1000) and chemiluminescent HRP substrate (Millipore) were used to detect the labeled bands. Pixel density was quantified with ImageJ (NIH) software.
The effects of nonsteroidal analogues of 2ME2BisMATE on cell viability depend on NF2 status
The STX therapeutic agents tested are multifaceted in their mechanisms of action. This study was directed at discovering whether the mechanisms of action that make the STX agents promising potential therapeutics for the treatment of NF1 would also apply to the potential treatment of NF2. Our previous studies concluded that the agents’ effects on the growth of and induction of apoptosis in human NF1 tumour cells were both through NF1-specific pathways and through significant effects on both the actin-based and myosin-based cytoskeleton . The same approaches in the present study on NF2 led to the findings that, in NF2 human tumour cells, none of the pathways that were found to be affected in NF1 was affected in NF2. However, effects on NF2 cell growth were also mediated through the STX agents’ effects on the cytoskeleton.
Next we investigated the mode of action of STX agents on the growth and/or induction of apoptosis in NF2 tumour cells and compared the effectiveness of these agents with those of Pak inhibitors. Our results, which will be discussed in the next section, indicated that STX agents caused depolymerization of microtubules, whilst Pak inhibitors did not have this effect.
Despite carefully repeated experiments (n = 5 [at least], on each of the cell lines) testing whether the STX agents act mechanistically on the same pathways on which we found them to be very effective on growth and apoptosis in human NF1 tumour cell lines, we currently have been able to detect NO significant effects of either of the agents on the growth, morphology of NF2 cell lines through the pathways that are known to be important for the growth of or cell death in NF1 human tumour cell lines (as detailed in Shen et al., 2015 ). This is an important point, because if the STX agents successfully reduce growth or induce apoptosis in NF2 cell lines as they do in NF1 cell lines, understanding the mechanism or mechanisms by which such effects occur is critical in order to plan the required preclinical animal studies that must follow the cell line studies if the agents are to be helpful eventually to both NF1 and NF2 patients.
STX3451 and STX2895 cause nuclear fragmentation in NF2-null tumour cells
Our previous studies on NF1 cell lines demonstrated that STX3451 caused nuclear fragmentation in both the NF1-null malignant tumour cell line ST88 and in benign plexiform neurofibroma (PNF) cells, although this result was less significant for PNF cells .
For Ben-Men-1 cells, the dramatic increase seen in nuclei fragmentation also fell between 24 and 48 h after treatment, with STX3451 slightly more effective than STX2895. Neither analog caused significant increase of fragmentation between 48 and 72 h, presumably due to the greatly reduced cell viability seen during this time (Fig. 2). However, the nonsteroidal sulfamate derivatives of 2ME2 effectively promoted nuclear fragmentation in both NF1  and NF2 null tumour cell lines.
STX3451 and STX2895, but not the Pak inhibitors, induce microtubular disruption; treatment with all agents resulted in tumour cell apoptosis
STX3451 induced apoptosis and disrupted both microtubule- and microfilament-based cytoskeletal structures in NF1 ST88 cells, but did not induce programmed cell death in the benign PNF tumour cells . To analyze apoptosis, we used an Annexin V antibody  to stain cells treated with STX agents and PAK inhibitors for 72 h. In healthy, quiescent cells phosphatidylserine (PS) is located exclusively at the inner cell membrane. Exposure of PS in a cell indicates early apoptosis . Annexin V has a high binding affinity for PS in the presence of Ca2+ and is therefore commonly used to detect apoptotic cells.
For Ben-Men-1 cells, although cell viability was little affected after 4 days of treatment, about 40% of these cells treated with either STX3451 or STX2895 (at 600 nM) were apoptotic at 72 h, indicating that these analogues of 2ME2 also have effects on cell growth/apoptosis in the benign Ben-Men-1 cells. However, the effects seen are much less than those seen when either STX agent was used to treat malignant KT21 cells. By contrast, 72 h treatment with PF resulted in 20% annexin V-positive cells at very low concentration, the IC50 for KT21 was 29.25 nM . Frax1036 at 1.9 μM caused 30% of Ben-Men-1 cells to become apoptotic at 72 h. Even though IOMM-Lee cell line viability was higher after 4 days, despite treatment with STX3451 or STX2895 at 1000 nM, annexin V-staining at 72 h demonstrated that 35 and 45% of these cells were undergoing cell death, respectively. The reason for this inconsistency is not clear. It could be that cells were still proliferating at earlier times, but eventually would be killed by higher concentrations of these 2ME2 analogs. Further studies are needed to determine the mechanism of action of these agents in IOMM-Lee cells. PF at 1.5 μM and Frax1036 at 10 μM resulted in apoptosis in about 25% of cells at 72 h, indicating that these agents are less effective in killing IOMM-Lee cells than either STX3451 or STX2895.
Finally, STX3451 and STX2895 at 1000 nM did not significantly increase the percentage of annexin V-positive BJ cells at 72 h, consistent with the results seen in the cell viability assays (discussed above). At concentrations that effectively induced apoptosis in NF2 tumour cells, STX3451 and STX2895 did not cause such programmed cell death in BJ fibroblasts. PF at 5 μM also did not increase the percentage of annexin V-positive BJ cells, whilst Frax1036 at 10 μM induced 55% cells to go through apoptosis.
STX3451 has been demonstrated to depolymerize microtubules in both NF1 ST88 and PNF cell lines . Our current studies also confirmed the effect of STX3451 and STX2895 on microtubules in NF2-null tumour cells. Both agents caused cells to round up and to present with much shorter microtubules in all of the cell lines examined (Fig. 5a). Failure of cytokinesis was also seen in these treated cells, where cells with larger diameters were observed (Fig. 5a). PF and Frax1036, in contrast to the STX agents, did not result in microtubule disruption in most of the cell lines tested. A high concentration of Frax1036 (10 μM) resulted in rosette-like clusters of IOMM-Lee cells. However, the mechanism for this rosette formation is unknown.
Our results from annexin V and α-tubulin staining (Fig. 5) suggest that the two analogs of 2ME2 that we evaluated act differently from the Pak inhibitors on NF2 cells, especially in the effects seen on the microtubular cytoskeleton. However, both STX agents and Pak inhibitors can promote apoptosis in NF2 tumour cells.
STX3451 and STX2895 decrease cell migration
We previously demonstrated that STX3451 induces apoptosis in human NF1-deficient ST88 and S462 malignant peripheral nerve sheath tumour (MPNST) cell lines at very low concentration (300 nM), whilst arresting cell proliferation in an NF1−/−benign plexiform neurofibroma (PNF) . In this study, using approaches and analyses identical to those we previously used in our NF1 tumour cell studies , we found that STX3451 and another potent sulfamoylated non-steroidal compound, STX2895, effectively induced cell death in NF2 deficient cell lines in vitro at concentrations between 0.3 μM and 1 μM. These two compounds have similar potencies in most of the NF2 cell lines, although STX2895 is slightly more potent than STX3451.
Intriguingly, our results showed that STX3451 and STX2895 were not as effective against a permanent NF2 wild type malignant meningioma cell line, IOMM-Lee, indicating that NF2 status could be important for the effects of these compounds. Our results also showed that these 2ME2 analogs were more potent than Pak inhibitors in inducing cell death. However, their mechanistic effects on the cells were different: STX3451 and STX2895 affect microtubules (Fig. 5), whilst Pak inhibitors act through the mTORC pathway and affect cell cycle progression by reducing β-catenin signaling, followed by reducing cyclin D1 expression . Further, Pak inhibitors did not affect microtubule polymerization at the concentrations we used. Therefore, the 2ME2 analogs provide a new means of treating NF2-related disorders, and could potentially be combined with Pak inhibitors in multi-drug approach treatments.
We also found that STX3451 and STX2895 caused nuclear fragmentation and apoptosis in NF2 deficient cells, as shown by annexin V staining. We found that by 72 h after treatment with STX3451, the percentage of annexin V positive cells amongst the attached cells was 19 times greater in KT21 and 4 times greater in Ben-Men-1 cells than in control cells. Treatment with STX2895 resulted in 20 times more annexin V positive KT21 cells and 4.5 times more Ben-Men-1 cells than that of the controls. STX3451 and STX2895 also increased the percentage of annexin V positive IOMM-Lee cells, but this increase was not statistically significant, indicating that NF2 wild-type tumour cells are less sensitive to the 2ME2 analogs. In addition, as was also shown in our studies of NF1-deficient cells , STX3451 caused microtubule depolymerization in NF2-deficient cells. STX2895 had very similar effects to those of STX3451 in NF2-deficient cells. These effects on the cytoskeleton undoubtedly contribute to the decrease in cell migration (Fig. 6) after treatment with the 2ME2 analogs, since disrupting microtubules can affect cellular locomotion . Both STX3451  and STX2895  have been shown to inhibit tubulin assembly in vitro, presumably by binding to the colchicine-binding site of tubulin. Indeed, our recent X-ray crystalographic study  has demonstrated, in atomic detail, that a non-steroidal quinazolinone sulfamate derivative, similar to those investigated here, can interact with the colchicine binding site and microtubule destabilization is likely achieved by preventing the curved-to-straight conformational transition in α/β-tubulin. This is the first atomic level demonstration of such an interaction for a sulfamate ester. Associated crystallographic work has also demonstrated the effectiveness of STX3451 in binding to the colchicine site .
Our previous study showed that the non-steroidal analog of 2ME2, STX3451, caused microtubule depolymerization and cell death in NF1 deficient tumour cells, even in the presence of elevated hormones . Whether steroid hormones affect the growth of NF2−/− vestibular schwannomas (VS) and/or meningiomas has not been well-studied. It has been shown that in sporadic VS, both oestrogen receptor (ER) and progesterone receptor (PR) were up-regulated, whilst in NF2-related VS, PR was down-regulated . Amongst meningioma patients, two-thirds express PR (although − 30% express PR at low levels), and during malignant progression, PR expression tends to decrease . PR-negative meningiomas also tend to be larger than those that are PR-positive , and expression of PR in meningiomas is correlated with a favourable prognosis . However, several studies have shown that hormone replacement therapy in postmenopausal women is associated with increasing the risk of meningiomas [60, 61] and one case report showed that cessation of long-term use of the PR agonist megestrol acetate resulted in shrinkage of multiple meningiomas in one patient . Treatment of progressive meningiomas with the PR antagonist mifepristone has not shown promising results . Therefore, hormone therapies are not indicated and may even be contraindicated for NF2-related tumours. The ability of STX3451 and STX2895 to inhibit colchicine binding to microtubules could play a major role in microtubule depolymerization and subsequent death of NF2 cells. However, the fact that, even at high concentration as 1 μM, BJ and IOMM-Lee cells still proliferate, although at a slower pace than the controls (Fig. 2), indicates that these small molecules could interact with players in the Merlin pathway in addition to their effects on microtubules. These possibilities need to be further investigated.
Since Merlin affects several key signalling pathways in the cytoplasm and nucleus, including PI3K signaling, mTORC1, the RAS and Src pathways, the Hippo kinase cascade, and CRL4-DCAF , inhibitors of these pathways might serve as excellent candidates for drug targets. Indeed, of the targeted drugs tested in clinics, bevacizumab, which is a VEGF antibody, showed some improvement in hearing and tumour shrinkage in a quarter to a half of patients with VS, albeit in very small numbers of study subjects [23, 24, 25]. Some phase II clinical trials with bevacizumab are still ongoing. The results of treatments with everolimus, an mTOR inhibitor, showed that it stabilized tumours or delayed tumour growth , but was ineffective for progressive VS . Lapatinib, a tyrosine kinase inhibitor for the epithelial growth factor receptor (EGFR), showed some success in reducing VS volumes and improving hearing responses in about one-quarter of a small group of patients in a phase II study , but no effect on meningiomas. Other phase II studies of this drug are also ongoing.
So far, none of the drugs tested in vitro or in pre-clinical or clinical trials has been effective on NF2 meningioma. Although, another mTOR inhibitor, AZD-2014, and a combination of smoothened receptor inhibitor, vismodegib, and FAK inhibitor, vistusertib, are in phase II trials. AR-42, a histone deacetylase inhibitor targeting CRL4-DCAF, which induced meningioma cell apoptosis in vitro and in xenografts, is also in phase I trials. Even if some inhibitors for NF2/Merlin signalling pathways have promising efficacy, drug resistance to these inhibitors may eventually occur and alternative choices are necessary. The microtubule-disrupting non-steroidal 2ME2 analogs could provide another option for treating NF2-related tumours, both vestibular Schwannomas and meningiomas, either alone, or in conjunction with other agents. Pre-clinical studies using several similar THIQ-based molecules have been shown to be safe and efficacious for reducing the size of human breast cancer  and melanoma xenografts [32, 45] in immunocompromised mice. Before testing the usefulness of STX3451 and STX2895 in human schwannoma and meningioma patients, pre-clinical studies are warranted. It is our hope that someday these small molecules can advance to therapeutic use in such patients.
Both STX3451 and STX2895 provide new approaches for inducing cell death and lowering tumour burden in NF2 as well as in NF1, which both have limited treatment options.
Support for this work was provided by the U.S. NSF: IOS1146132 and U.S. NIH: R25GM086262 to KFB; U.S. NIH: R01 CA148805 to JC, and the Wellcome Trust Senior Investigator grant to BVLP (grant 101010).
YS, CA, RM, BVLP and KFB were involved in the conception and design of the study, analyzing and interpreting data, writing the manuscript and revision of the final version. EMJ designed and performed statistical analyses. YS, CA and RM carried out experiments including cell culture and proliferation, IHC, migration, and invasion assays. WD and BVLP provided the synthetic compounds. WD, SK, JC, EMJ and BVLP contributed in writing the draft and revisions. All authors have read and approved the manuscript.
Support for this work was provided by the U.S. NSF: IOS1146132 and U.S. NIH: R25GM086262 to KFB; U.S. NIH: R01 CA148805 to JC and the Wellcome Trust to BVLP. BVLP is a Wellcome Trust Senior Investigator (grant 101010).
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
- 14.Yi C, Troutman S, Fera D, Stemmer-Rachamimov A, Avila JL, Christian N, Persson NL, Shimono A, Speicher DW, Marmorstein R, et al. A tight junction-associated Merlin-angiomotin complex mediates Merlin's regulation of mitogenic signaling and tumor suppressive functions. Cancer Cell. 2011;19:527–40.PubMedPubMedCentralCrossRefGoogle Scholar
- 18.Roger E. McLendon, Marc K. Rosenblum, Darell D. Bigner. Neurofibromatosis 2. Russell & Rubinstein’s Pathology of Tumors of the Nervous System 7Ed 2006;p917–927.Google Scholar
- 22.Karajannis MA, Legault G, Hagiwara M, Ballas MS, Brown K, Nusbaum AO, Hochman T, Goldberg JD, Koch KM, Golfinos JG, et al. Phase II trial of lapatinib in adult and pediatric patients with neurofibromatosis type 2 and progressive vestibular schwannomas. Neuro-Oncology. 2012;14:1163–70.PubMedPubMedCentralCrossRefGoogle Scholar
- 25.Blakeley JO, Ye X, Duda DG, Halpin CF, Bergner AL, Muzikansky A, Merker VL, Gerstner ER, Fayad LM, Ahlawat S, et al. Efficacy and biomarker study of Bevacizumab for hearing loss resulting from Neurofibromatosis type 2-associated vestibular Schwannomas. J Clin Oncol. 2016;34:1669–75.PubMedPubMedCentralCrossRefGoogle Scholar
- 26.Goutagny S, Raymond E, Esposito-Farese M, Trunet S, Mawrin C, Bernardeschi D, Larroque B, Sterkers O, Giovannini M, Kalamarides M. Phase II study of mTORC1 inhibition by everolimus in neurofibromatosis type 2 patients with growing vestibular schwannomas. J Neuro-Oncol. 2015;122:313–20.CrossRefGoogle Scholar
- 28.Bush ML, Oblinger J, Brendel V, Santarelli G, Huang J, Akhmametyeva EM, Burns SS, Wheeler J, Davis J, Yates CW, et al. AR42, a novel histone deacetylase inhibitor, as a potential therapy for vestibular schwannomas and meningiomas. Neuro-Oncology. 2011;13:983–99.PubMedPubMedCentralCrossRefGoogle Scholar
- 30.Shen YC, Upadhyayula R, Cevallos S, Messick RJ, Hsia T, Leese MP, Jewett DM, Ferrer-Torres D, Roth TM, Dohle W, Potter BVL, Barald KF. Targeted NF1 cancer therapeutics with multiple modes of action: small molecule hormone-like agents resembling the natural anticancer metabolite, 2-methoxyoestradiol. Br J Cancer. 2015;113:1158–67.PubMedPubMedCentralCrossRefGoogle Scholar
- 48.Roth TM, Ramamurthy P, Muir D, Wallace M, Zhu Y, Chang L, Barald KF. Influence of hormones and hormone metabolites on the growth of Schwann cells derived from embryonic stem cells and on tumor cell lines expressing variable levels of neurofibromin. Dev Dyn. 2008;237:513–24.PubMedCrossRefGoogle Scholar
- 54.Dohle W, Jourdan FL, Menchon G, Prota AE, Foster PA, Mannion P, Hamel E, Thomas MP, Kasprzyk PG, Ferrandis E, et al. Quinazolinone-based anticancer agents: synthesis, Antiproliferative SAR, Antitubulin activity, and tubulin co-crystal structure. J Med Chem. 2018;61:1031–44.PubMedCrossRefGoogle Scholar
- 55.Dohle W, Prota AE, Menchon G, Hamel E, Steinmetz MO, Potter BVL. Tetrahydroisoquinoline Sulfamates as potent microtubule disruptors: synthesis, anti-proliferative and anti-tubulin activity of Dichlorobenzyl derivatives and a tubulin co-crystal structure. ACS Omega. 2019;4:755–64.PubMedPubMedCentralCrossRefGoogle Scholar
- 65.Karajannis MA, Legault G, Hagiwara M, Giancotti FG, Filatov A, Derman A, Hochman T, Goldberg JD, Vega E, Wisoff JH, et al. Phase II study of everolimus in children and adults with neurofibromatosis type 2 and progressive vestibular schwannomas. Neuro-Oncology. 2014;16:292–7.PubMedCrossRefGoogle Scholar
- 66.Stengel C, Newman SP, Day JM, Chander SK, Jourdan FL, Leese MP, Ferrandis E, Regis-Lydi S, Potter BVL, Reed MJ, Purohit A, Foster PA. In vivo and in vitro properties of STX2484: a novel non-steroidal anti-cancer compound active in taxane-resistant breast cancer. Br J Cancer. 2014;111:300–8.PubMedPubMedCentralCrossRefGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.