Patent Application
20200093835
The present invention relates to methods to reduce the proliferation of vestibular schwannoma cells. The methods include administering mifepristone.
Vestibular schwannoma (VS) is the fourth most common intracranial tumor and the most common tumor of the cerebellopontine angle, arising from neoplastic Schwann cells of the vestibular nerve. No drug is FDA-approved to treat VS. In 95% of patients, these tumors cause debilitating sensorineural hearing loss (SNHL) and tinnitus and often lead to dizziness and facial paralysis. Bilateral VSs are the hallmark of neurofibromatosis type 2 (NF2), an autosomal dominant disorder caused by inactivation or loss of both alleles of the NF2 gene. If left untreated, growing VSs can compress the brainstem and lead to death. Currently, patients with symptomatic or growing VSs can undergo surgical resection or radiotherapy, both procedures that can result in serious complications.
Bilateral VSs are the hallmark of neurofibromatosis type 2 (NF2), an autosomal dominant disorder caused by inactivation or loss of both alleles of the NF2 gene. Mutations in the NF2 gene are identified in 100% of NF2-associated VSs and 66% of sporadically arising VSs6,7. Though mechanisms of VS-induced SNHL are multifactorial, with contributions from tumor size, localized or systemic infection, inflammation, and tumor-secreted factors8,9, NF2-associated SNHL often correlates with VS size8,10. This observation suggests that slowing or inhibiting VS growth may not only prolong a patient's time to surgical intervention, but also minimize or prevent associated SNHL, substantially improving quality of life.
Using publicly available omics data to probe relationships between genes, small molecules, and disease, the computational repositioning of existing drugs represents an appealing avenue for identifying potentially effective compounds, particularly for diseases with no FDA-approved pharmacotherapies. Here we present the first application of algorithm-based drug repositioning to neuro-otology, culminating in the computational repositioning and preclinical validation of mifepristone for human vestibular schwannoma (VS), a debilitating intracranial tumor. We applied ksRepo, an open-source computational drug repositioning platform3, to the largest meta-analysis of transcriptomic data from human VS patients, identifying eight promising drugs approved by the FDA with potential for repurposing in VS. Of these eight, we showed that mifepristone, a progesterone and glucocorticoid receptor antagonist, adversely affects the morphology, metabolic activity, and proliferation of HEI-193 human schwannoma cells, as well as that of primary human VS cells. Mifepristone treatment produces a more dramatic reduction in the metabolic activity of primary human VS cells than cells derived from patient meningiomas, while primary human Schwann cells remain unaffected.
Thus, provided herein are methods for reducing the proliferation of a vestibular schwannoma cell, wherein the method comprises contacting the vestibular schwannoma cell with an effective concentration of mifepristone. In some embodiments, the vestibular schwannoma cell is ex vivo; in some embodiments, the vestibular schwannoma cell is in a subject, e.g., a mammal, e.g., a human.
In some embodiments, the subject is or has been diagnosed as having vestibular schwannoma, e.g., using methods known in the art.
Also provided herein are methods for treating a subject having vestibular schwannoma. The methods include administering to the subject a therapeutically effective amount of mifepristone.
Further provided herein are methods for reducing the rate of vestibular schwannoma tumor growth in a subject that include administering to the subject a therapeutically effective amount of mifepristone.
In addition, provided herein are methods for inducing or increasing vestibular schwannoma cell death in a subject in need thereof that include administering to a subject a therapeutically effective amount of mifepristone.
In some embodiments of the methods described herein, the administration is local administration, e.g., by injection through the ear drum, or direct delivery into the inner ear.
In some embodiments of the methods described herein, the administration is systemic administration.
In some embodiments of the methods described herein, the systemic administration is oral, intravenous, intraarterial, nasal, intramuscular, subcutaneous, or intraperitoneal administration.
In some embodiments of the methods described herein, the subject has been diagnosed as having vestibular schwannoma.
In some embodiments of the methods described herein, the methods include a step of identifying or diagnosing a subject as having vestibular schwannoma.
The present methods can also be used in subjects having closely related schwannomas arising from other cranial nerves, such as schwannoma of the oculomotor, trigeminal, facial, hypoglossal, or vagal nerves within or outside of the parapharyngeal space and cutaneous schwannomas.
As used herein, the word “a” before a noun represents one or more of the particular noun. For example, the phrase “a vestibular schwannoma cell” represents “one or more vestibular schwannoma cells.”
The term “subject” means a vertebrate, including any member of the class mammalia, including humans, rats, mice, rabbits, sports or pet animals, such as horse (e.g., race horse) or dog (e.g., race dogs), and higher primates. In preferred embodiments, the subject is a human.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
Vestibular schwannomas (VSs), the most common tumors of the cerebellopontine angle, can cause substantial morbidity. There is a clinical need to develop pharmacotherapies against VS as current treatments carry significant risks. Described herein are specific pathways involved in the pathobiology of neoplastic VS growth and VS-associated SNHL and therapeutic targets that regulate neoplastic VS growth and VS-induced SNHL.
Clinical Features and Incidence of Vestibular Schwannomas (VSs)
Neoplastic Schwann cells (SCs) of the vestibular nerve lead to VSs, the fourth most common intracranial tumors. VSs, although benign in nature, can lead to various symptoms due to their crucial location within the internal auditory canal that houses the vestibulocochlear and facial nerves (Tew & McMohan, 2013). Ninety-five percent of VS patients suffer from sensorineural hearing loss (SNHL), with a smaller percentage suffering from vestibular dysfunction and facial nerve paralysis (Matthies & Samii, 1997). Further, due to their expansion into the cerebellopontine angle, VSs can lead to brainstem compression and death as the tumors grow larger (Charabi et al., 2000).
To alleviate this tumor burden, patients can undergo surgical resection or stereotactic radiotherapy. Surgical resection entails full or partial removal of the tumor via craniotomy and carries substantial risks, including SNHL, vestibular dysfunction, facial nerve paralysis, cerebrospinal fluid leaks and meningitis (Sughrue et al., 2011a; Mahboubi et al., 2014). Stereotactic radiotherapy entails delivering a radiation dose to the tumor and also carries substantial risks such as further exacerbation of the SNHL, vestibular dysfunction and malignant transformation of the tumor (Demetriades et al., 2010; Collens et al., 2011). Patients with non-growing or asymptomatic VSs can undergo conservative management and follow the tumor's progression through serial magnetic resonance imaging (MRI), but due to the lack of biomarkers for VS growth and associated symptoms, it can be a risky approach (Thakur et al., 2012). Reliable biomarkers and effective drug therapies would greatly advance health care for VS patients. In this disclosure, with an eye towards identifying effective biomarkers and pharmacotherapies, several pathobiological pathways in VS growth and VS associated SNHL were investigated.
Clinical incidence of VS has been approximately 19 per million per year (Stangerup & Cayé-Thomasen, 2012). The first VS and associated SNHL were described in 1830 by Sir Charles Bell and incidence rates have increased considerably over time, partially attributed to the advent of imaging. Although cell phone radiation-induced neoplastic transformation has been postulated, most studies investigating correlation of cell phone use with VS incidence show negative findings (Pettersson et al., 2014). Interestingly, histologic incidence for VS is approximately 1 per 500, as assessed through MRIs conducted on a group of 2000 subjects from the general population (Vemooij et al., 2007). Further, the vestibular nerve serves as a predilection site for schwannomas, with 57% of schwannomas occurring on this nerve (Propp et al., 2006). These unusually high incidence rates suggest an intriguing biology of the vestibular nerve and VS.
Methods of Treatment
The methods described herein can be used to treat subjects with VS, e.g., subjects who have been diagnosed with VS, or having closely related schwannomas arising from other cranial nerves, such as schwannoma of the oculomotor, trigeminal, facial, hypoglossal, or vagal nerves within or outside of the parapharyngeal space and cutaneous schwannomas. The methods include administering a therapeutically effective concentration of mifepristone. Mifepristone (RU486), 11β-(4-dimethylaminophenyl)-17β-hydroxy-17α-(1-propynyl)-estra-4,9-dien-3-one, is a progesterone and glucocorticoid receptor antagonist currently approved by the FDA for use in medical abortion. This steroid analog is able to cross the blood-brain barrier12 and has been shown in human clinical trials to provide palliative benefits to patients with other intracranial tumors, such as glioblastoma multiforme12 and meningioma13,14. In vitro, mifepristone produces antiproliferative effects on cervical15,16, breast17,18, endometriall9,20, ovarian21,22, gastric23, bile duct24, and prostate cancer cells25,26, regardless of progesterone receptor expression27. In human trials, mifepristone administration has been documented to significantly improve quality of life for patients suffering from advanced thymic, renal, colon, leukemic, and pancreatic cancers28,29. Long-term administration of oral mifepristone is well tolerated by adults and carries only a mild toxicity profile13.
In some embodiments, subjects treated with the present methods do not have, or have not been diagnosed with, a neurofibroma; in some embodiments, they have (or have been diagnosed with) a multiple schwannoma disorder, e.g., neurofibromatosis type 2, schwannomatosis, or Carney complex (See, e.g., Rodriguez et al., Acta neuropathologica. 2012; 123(3):295-319). In some embodiments, subjects treated with the present methods have bilateral VSs (and not neurofibromas), and may have neurofibromatosis type 2 (NF2).
There is no consensus about whether VS tumors even express PR. In 2006, an immunohistochemical study of 100 VSs found that no tumors expressed the PR (Jasiwal et al., Journal of Negative Results in Biomedicine. 2009; 8:9). In 2008, a similar immunohistochemical study of 59 tumors found that all tumors expressed the PR (Caifer et al., J Laryngol Otol. 2008 February; 122(2): 125-7). The same year, Dalgorf et al. showed that nine of nine tested VSs were “unequivocally negative” for PR (Dalgorf et al., Skull Base. 2008; 18(6):377-384). Later that year, Patel et al. claimed that 16 of 23 sporadic VSs upregulated PR messenger RNA (mRNA) expression, but that NF2-associated VSs significantly downregulated PR mRNA expression (Patel et al., The Laryngoscope. 2008; 118(8):1458-1463). Therefore, with no clear consensus in the field regarding PR expression in VS, McLaughlin et al.'s claim cannot be considered applicable to this tumor. In our own meta-analysis comprising 80 VSs, PR mRNA was concordantly downregulated (false discovery rate p=0.01). When testing mifepristone on primary human VS cells, we observed no correlation between response to mifepristone and PR expression. In some embodiments, the subject does not have a progesterone receptor (PR)-expressing VS; in these embodiments, the methods can include determining whether the VS expresses PR (see, e.g., WO2004010928), and excluding those that do express PR in the schwannoma cells.
In some embodiments, the methods include determining that the subject is not pregnant or not likely to become pregnant.
Pharmaceutical Compositions
The methods described herein can include administration of mifepristone as an active agent in a pharmaceutical composition. Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
The present pharmaceutical compositions are formulated to be compatible with the intended route of administration.
In some embodiments, the compositions are delivered systemically, e.g., by oral, parenteral, e.g., intravenous, intradermal, or subcutaneous administration.
In some embodiments, the compositions are administered by local administration to the vestibular schwannoma, e.g., by application of a liquid, foam, or gel formulation to the round window membrane. Application to the round window membrane can be accomplished using methods known in the art, e.g., intra-tympanic injection of a liquid, foam, or gel formulation or by direct delivery into the inner ear fluids, e.g., using a microfluidic device such as an implantable pump.
Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, N.Y.). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
In some embodiments, the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies to cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811. Nanoparticles, e.g., poly lactic/glycolic acid (PLGA) nanoparticles (see Tamura et al., Laryngoscope. 2005 November; 115(11):2000-5; Ge et al., Otolaryngol Head Neck Surg. 2007 October; 137(4):619-23; Horie et al., Laryngoscope. 2010 February; 120(2):377-83; Sakamoto et al., Acta Otolaryngol Suppl. 2010 November; (563):101-4) can also be used.
In some embodiments, the carrier comprises a polymer, e.g., a hydrogel, that increases retention of the compound on the round window and provides local and sustained release of the active ingredient. Such polymers and hydrogels are known in the art, see, e.g., Paulson et al., Laryngoscope. 2008 April; 118(4):706-11 (describing a chitosan-glycerophosphate (CGP)-hydrogel based drug delivery system); other carriers can include thermo-reversible triblock copolymer poloxamer 407 (see, e.g., Wang et al., Audiol Neurootol. 2009; 14(6):393-401. Epub 2009 Nov. 16, and Wang et al., Laryngoscope. 2011 February; 121(2):385-91); poloxamer-based hydrogels such as the one used in OTO-104 (see, e.g., GB2459910; Wang et al., Audiol Neurotol 2009; 14:393-401; and Piu et al., Otol Neurotol. 2011 January; 32(1): 171-9); Pluronic F-127 (see, e.g., Escobar-Chavez et al., J Pharm Pharm Sci. 2006; 9(3):339-5); Pluronic F68, F88, or F108; polyoxyethylene-polyoxypropylene triblock copolymer (e.g., a polymer composed of polyoxypropylene and polyoxyethylene, of general formula E106 P70 E106; see GB2459910, US20110319377 and US20100273864); MPEG-PCL diblock copolymers (Hyun et al., Biomacromolecules. 2007 April; 8(4):1093-100. Epub 2007 Feb. 28); hyaluronic acid hydrogels (Borden et al., Audiol Neurootol. 2011; 16(1):1-11); foams, e.g., as described in WO2009132050A9, WO2011049958A2, WO2015031393A1, or WO2010048095A2; gelfoam cubes (see, e.g., Havenith et al., Hearing Research, February 2011; 272(1-2):168-177); and gelatin hydrogels (see, e.g., Inaoka et al., Acta Otolaryngol. 2009 April; 129(4):453-7); other biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Tunable self-assembling hydrogels made from natural amino acids L and D can also be used, e.g., as described in Hauser et al e.g. Ac-LD6-COOH (L) e.g. Biotechnol Adv. 2012 May-June; 30(3):593-603. Such formulations can be prepared using standard techniques, or obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. In some embodiments, the composition (e.g., in foam or gel form) is applied to the tympanic membrane, e.g., as described in WO2009132050A9, WO2011049958A2, WO2015031393A1, or WO2010048095A2.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
Dosage
An “effective amount” is an amount sufficient to effect beneficial or desired therapeutic effect. This amount can be the same or different from a prophylactically effective amount, which is an amount necessary to prevent onset of disease or disease symptoms. An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a therapeutic compound (i.e., an effective dosage) depends on the therapeutic compounds selected. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a single treatment or a series of treatments. In some embodiments, e.g., in subjects exposed to prolonged or repeated exposures to noise, e.g., normal noises such as are associated with activities of daily life (such as lawnmowers, trucks, motorcycles, airplanes, music (e.g., from personal listening devices), sporting events, etc.), or loud noises, e.g., at concert venues, airports, and construction areas, that can cause inner ear damage and subsequent hearing loss; e.g., subjects who are subjected to high levels of environmental noise, e.g., in the home or workplace, can be treated with repeated, e.g., periodic, doses of the pharmaceutical compositions, e.g., to prevent (reduce the risk of) or delay progression or hearing loss.
Dosage, toxicity and therapeutic efficacy of the therapeutic compounds can be determined by standard pharmaceutical procedures, e.g., in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. For example, samples of the perilymph or endolymph can be obtained to evaluate pharmacokinetics and approximate an effective dosage, e.g., in animal models, e.g., after administration to the round window. The dosage of such compounds lies preferably within a range of concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated from cell culture assays, and/or a dose may be formulated in animal models; alternatively, for those compounds that have been previously used in humans, clinically desirable concentrations can be used as a starting point. Such information can be used to more accurately determine useful doses in humans.
In some embodiments, the dose is about 100-300 mg/day, e.g., about 200 mg/day, delivered orally.
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
Methods
The following materials and methods were used in the Examples below.
GEO Dataset Processing
The GEO dataset used in this study is GSE39645, an Affymetrix Human Gene 1.0 ST chip-based gene expression study of VS which contained data from 28 patients with sporadic VS, 3 patients with NF2-associated VS, and 8 control nerve samples41. Data for GSE39645 was accessed through the NCBI GEO portal and analyzed using the integrated GEO2R tool42. As input for GEO2R, we classified each sample within a GEO series as either normal tissue or VS tissue. The GEO2R analysis was performed on both the full dataset (sporadic and NF2 combined), and a subset of samples containing only NF2-syndromic schwannomas. GEO2R provides a list of probes and corresponding gene symbols ranked according to their degree of differential expression (as calculated using the limma package in R43), and includes p-values and t-statistics for differential expression. Following GEO2R analysis, all results were imported into R44 and probe-level differential expression was consolidated to gene-level differential expression using a custom pipeline: t-statistic values were converted to Cohen's d statistic values and standard error values45. Resulting values were combined by gene using a fixed effects meta-analysis (as implemented in the meta.summaries function from the rmeta package in R46. Probes without gene annotations were removed from gene-level consolidation. Following consolidation47, significantly differentially expressed genes were taken to be those with a Bonferroni-corrected significance of less than 0.05.
Additional VS Dataset Processing
Raw Affymetrix Human Genome U219 gene expression data (.CEL files) for 36 patients with sporadic VS, 13 patients with NF2 syndrome-associated VS, and 7 control nerves were generously donated by Agnihotri et al.6. CEL files were loaded into R using the justRMA function from the affy package in R48. justRMA is an automated tool that both performs normalization using the Robust Multi-Array Average method49 and also automatically annotates all probes in the normalized dataset using the Org.Hs.eg.db annotation database package50. Normalization was performed on the full dataset and the NF2-associated schwannomas, as above. Mirroring the GEO2R analysis, each normalized dataset was analyzed using limma and consolidated to gene-level differential expression using the custom pipeline described above. As above, significantly differentially expressed genes were taken to be those with a Bonferroni-corrected significance of less than 0.05.
Meta-Analysis of 80 VS Samples and ksRepo Prediction
To robustly determine differential expression between VS and normal tissues, gene-level data from GSE39645 and Agnihotri et al.6 were meta-analyzed by first removing genes that were not measured in both the Affymetrix Human Gene 1.0 ST chip and the Affymetrix Human Genome U219 chip, and subsequently combining Cohen's d and standard error values using a fixed-effects meta-analysis (again using meta.summaries). Meta-analysis was performed for the full GSE39645 and Agnihotri datasets, as well as for NF2-associated tumors exclusively. Following meta-analysis, the remaining genes were ranked according to their meta-analytic p-values to generate a gene list for further analysis using ksRepo (package available for download at github.com/adam-sam-brown/ksRepo, and described in Brown et al (2016)3. ksRepo is a gene-based drug repositioning method that uses a modified Kolmogorov-Smimov (KS) statistic to identify promising drug repositioning opportunities. ksRepo requires a database of compound-gene interactions, which are compared with the ranked meta-analytic gene lists from above. For this analysis, the ksRepo built-in Comparative Toxicogenomics Database (CTD) dataset was selected. The CTD provides a curated resource that links small chemical entities to genes (e.g., gene or protein expression influences) from the scientific literature on numerous model organisms and humans11. ksRepo contains a subset of the CTD, containing human-derived interactions between 1,268 unique drugs and 18,041 unique human genes. Drugs in the CTD subset were chosen based on case-insensitive matches between CTD names and names/synonyms for FDA-approved drugs downloaded from DrugBank51. The ksRepo output provides both the resampled p-value and FDR value. For the full dataset ksRepo analysis and the NF2-only ksRepo analysis, significant compounds were those for which the FDR was less than 0.05.
Human Specimen Collection and Primary Cell Culture
Surgical VS and GAN specimens were collected and processed according to protocols approved by the Human Studies Committee of Massachusetts General Hospital and Massachusetts Eye and Ear (Board Reference #14-148H). Written informed consent was obtained from all subjects prior to inclusion in this study and all procedures were conducted in accordance with the Helsinki Declaration of 1975. Detailed methods for human surgical specimen collection, processing, and culture are previously published38. VS specimens were harvested from patients undergoing surgical tumor resection, and GAN specimens from healthy patients undergoing benign parotidectomy or neck dissection surgery, during which the GAN is routinely sacrificed. Patients who had received radiation therapy prior to surgery were excluded.
Briefly, after surgical resection, VS or GAN tissue was immediately placed in saline solution and transported to the laboratory on ice. Specimens were rinsed with Hank's Balanced Salt Solution (HBSS, ThermoFisher Scientific), dissected to remove burned tissue and blood vessels, and separated for RNA preservation (RNALater, ThermoFisher Scientific) or primary cell culture. After enzymatic dissolution (collagenase type I, 160 U/mL; hyaluronidase type I-S, 250 U/mL) and trituration with an 18-gauge needle, primary cell culture suspensions were plated on 12 mm coverslips pre-coated with poly-D-lysine and laminin (Neuvitro) and grown in Dulbecco's Modified Eagle's Medium (DMEM) and F12-containing medium (ThermoFisher Scientific) consisting of 44.5% DMEM, 44.5% F12 nutrient mixture, 10% fetal bovine serum (ThermoFisher Scientific), and 1% of a mixture of penicillin and streptomycin (ThermoFisher Scientific). VS and GAN cultures were incubated at 37 degrees Celsius with 5% carbon dioxide, and culture medium was changed every three days. All downstream procedures were performed on primary cell cultures or collected culture medium at two weeks of age in culture to ensure maximal Schwann or schwannoma cell purity38.
HEI-193 and Arachnoid Cell Culture
HEI-193 cells are derived from a patient with sporadic bilateral vestibular schwannomas and a history of meningioma; these cells express a splice variant of the merlin protein (encoded by the NF2 gene), but neither typical isoform52. HEI-193 cells were cultured in DMEM/F12-containing medium with 10% fetal bovine serum and 1% penicillin and streptomycin mix as described above. Immortalized NF2-null and NF2-expressing arachnoid AC-CRISPR cell lines derived from primary human autopsy specimens were obtained via generous gift from Dr. Vijaya Ramesh at Massachusetts General Hospital40. NF2-null and NF2-expressing arachnoid cells were cultured in DMEM with 15% fetal bovine serum and 1% penicillin and streptomycin mix. All cell lines were maintained in an incubator at 37 degrees Celsius with 5% carbon dioxide and treated with drugs 24-36 hours after seeding at between 15,000-25,000 cells per well in 24-well plates. Phase contrast photos of healthy and drug-treated cultures were taken at 10× magnification on an IncuCyte S3 instrument (Essen Bio).
Drug Preparation and Treatment
Primary VS and GAN cultures were treated with mifepristone (Sigma Aldrich, lot # WXBC0031V) and progesterone (Sigma Aldrich, lot # SLBQ9723V). Fifteen, 25, 35, and 70 μM mifepristone, and 35 μM progesterone were prepared by suspending the appropriate amount of drug (in powder form) in dimethyl sulfoxide (DMSO). The resulting drug suspension was diluted in culture medium to the concentration of interest, and drug-containing medium was applied to primary VS, GAN, and HEI-193 cells such that the amount of DMSO applied to cells in culture did not exceed 0.1% (24-well plate, 1 mL medium per well). Cultures were incubated with drug-containing medium or 0.1% DMSO vehicle for 72 hours and then processed for downstream applications.
Proliferation Assay
5′bromo-2′-deoxyuridine (BrdU) was added to label proliferating cells in culture 2 hours before fixation in 4% formalin (paraformaldehyde). Cell membranes were permeabilized with 10 minutes of incubation in 1% Triton X-100 and nuclear membranes with 20 minutes in 2N hydrochloric acid (HCl). Cells were blocked in 5% normal horse serum (NHS) and 1% Triton X-100 and incubated with a primary antibody against BrdU (# OBT0030G, AbD Serotec) overnight, followed by incubation with fluorescent anti-rat immunoglobulin G (AlexaFluor, Life Technologies). Cells were stained with Hoechst 33342 (Invitrogen) and phalloidin/f-actin (ThermoFisher Scientific) and coverslips mounted on slides with VectaShield (Vector Laboratories). The ratio of BrdU-positive to Hoechst-positive nuclei was determined by sampling three random fields of view using a Leica epifluorescence microscope. Manual counts were performed by J.E.S., who was blinded to treatment conditions by receiving and quantifying image files labeled only with arbitrary numbers and presented in random order.
Metabolic Activity Assay
The metabolic activity of primary VS and HEI-193 cells was assessed using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Life Technologies). After 72-hour incubation of cells in a 24-well plate with medium containing drug or vehicle, culture medium was replaced with 362 μL of colorless DMEM and 38 μL of 12 mM MTT in 3-6 random wells. Cells were incubated for an additional 4 hours. The resulting formazan crystals were dissolved in 380 μL of a solution of 100 mg/mL sodium dodecyl sulfate in 0.01 M HCl and incubated for another 4 hours. Optical density (OD) at 570 nm of each well was detected using a spectrophotometer. The average OD value of cells exposed to vehicle (0.1% DMSO) was set to 100% and used to normalize OD values of cells treated with drugs; metabolic activity was then reported as percent change. Statistical testing was performed on raw OD values (see Statistical Methods).
Flow Cytometry
Apoptotic cell death was assessed using an Annexin V/propidium iodide (PI) staining kit (Miltenyi Biotec). Briefly, HEI-193 cells were seeded into T25 flasks and treated with mifepristone or DMSO vehicle in culture medium for 24 hours as described above. Adherent cells were collected by trypsinization, and non-adherent (floating) cells were collected from culture medium. Cells were centrifuged, washed in PBS, and incubated in 1× annexin binding buffer, annexin V-fluorescein isothiocyanate (FITC), and propidium iodide (PI) according to the manufacturer's recommendations. Stained cells were immediately analyzed using a Cytomics FC500 flow cytometer. Data were analyzed using CXP Analysis software (version 2.2, Coulter).
Cell Cycle Analysis
Harvested HEI-193 cells were washed in PBS and fixed in cold 70% ethanol at −20° C. for 18-72 h. Before staining with propidium iodide (PI), cells were centrifuged and washed again in cold PBS. 2×106 or fewer cells were incubated with 500 μl staining solution [0.1% Triton X-100 (Sigma), 2 mg/ml RNase A (Qiagen), and 1 μg/ml PI (Miltenyi Biotec) in PBS] for 15 minutes at 37° C. In order to exclude DNAse activity, RNase A was boiled for 5 minutes and cooled down before its addition to staining solution. Cells were analyzed on a Cytomics FC500 flow cytometer using CXP Analysis software (version 2.2, Coulter).
Enzyme-Linked Immunosorbent Assay
Cell-conditioned medium was collected from mifepristone-treated and untreated HEI-193 cells after 72-hour incubation with the drug. Enzyme-linked immunosorbent assay (ELISA) was performed on each sample in triplicate to assess the quantity of progesterone in cell-conditioned medium, according to the manufacturer's protocol (Enzo Life Science, # ADI-900-011). Data were analyzed using GraphPad Prism 7 software licensed through Harvard Medical School.
Cytotoxicity and Cell Confluence Assays
Cytotoxicity and cell confluence were measured using live-cell, time-lapse phase contrast and fluorescence imaging acquired at 10× by an IncuCyte S3 instrument (Essen Bioscience). Cytotoxicity was measured by incorporation of IncuCyte Cytotox Reagent (Essen Bioscience), applied according to manufacturer's instructions; the reagent fluoresces when it binds DNA after compromise of membrane integrity. Nine images were acquired per well every 2 hours for 72 hours, and cytotoxicity at each time point is reported as number of fluorescent objects per well, after thresholding to avoid the inclusion of small cellular debris. Phase object confluence was measured using time-lapse phase contrast imaging acquired at 10× by the same instrument, analyzing 9 images per well every 2 hours for 72 hours, and is reported as percent confluence per square millimeter.
Ingenuity Pathway Analysis
Ingenuity Pathway Analysis software (Qiagen) was used to perform standard Core Analysis on all genes in our meta-analysis that reached significance (p<0.05) after Bonferroni correction for multiple hypothesis testing. Relevant upstream regulators for the resulting networks were identified and analyzed using published Ingenuity Pathway Analysis protocols (Qiagen).
gDNA Extraction
Genomic DNA (gDNA) was extracted from six vestibular schwannoma tissue samples using the DNeasy Blood and Tissue Kit (Qiagen) following manufacturer's specifications. The concentration of double-stranded DNA in each sample was evaluated using a Qubit dsDNA BR Assay Kit. A minimum measurement of 50 ng/μl was required for each sample to be included with HaloPlex target enrichment.
Library Preparation and Targeted Capture
A library of DNA restriction fragments from all coding exons, introns, and UTRs (5′ and 3′) of the NF2 gene was prepared using a HaloPlex HS target enrichment kit (Agilent Technologies), following the manufacturer's instructions. The total region size was 95.045 kbp with an actual analyzed target of 89.408 kbp bases, which required 2581 amplicons to achieve this 94.07% coverage with maximum validation stringency. Enrichment was performed according to the supplier's protocol by the Ocular Genomics Institute at Massachusetts Eye and Ear (Boston, Mass., USA).
Briefly, 50 ng of genomic DNA from each sample diluted with nuclease-free water to a final concentration of 1.8 ng/L were digested in eight different reactions, each containing two restriction enzymes. Successful digestion of ECD gDNA was indicated by the appearance of three predominant bands at 124, 255, and 450 bp, corresponding to the 800-bp PCR product-derived restriction fragments. A library of HaloPlex probes designed using the HaloPlex SureDesign program (www.genomics.agilent.com) was hybridized to the library of genomic DNA restriction fragments. Enrichment was validated by gel electrophoresis. Following purification, the DNA concentration of each library was quantified using the high-sensitivity D1000 DNA Tapescreen analysis assay on the Tapestation 2200 instrument (Agilent Technologies), and samples were subsequently sequenced.
NF2 Gene Sequencing
Targeted enrichment sample sequencing was performed on an Illumina MiSeq NGS platform (Illumina, Inc.) by the Next Generation Sequencing Core of the Massachusetts Eye and Ear Ocular Genomics Institute. The purified and individually tagged amplicon libraries for each sample were pooled equimolarly, and a percentage of an internal control (ECD) was added to validate the DNA sequencing and to help balance the overall lack of sequence diversity. The sample pool was then placed in a MiSeq Reagent kit version 2 500-cycle cartridge (Illumina) containing sequencing reagents, and sequencing was performed on the Illumina MiSeq instrument by using a MiSeq Reagent Kit v2 flow cell (Illumina). The quality criteria for MiSeq includes a number of generated clusters between 600 and 1200 K/mm2, >90% passed filter clusters, and approximately 5% sequenced ECD. To be included in the analysis, bases had at least a quality score of 40, and depth of coverage was at least 100 for all samples.
Bioinformatic Processing and Variant Prioritization
Raw data were demultiplexed and converted to fastq using Illumina bcl2fastq conversion software (v 2.16.0.10) as directed Agilent and Illumina. Prior to alignment, Agilent AGeNT (v3.5.1.46) was used to trim low-quality bases from the ends, remove adaptor sequences, and remove duplicated reads based on Molecular Barcode information following Agilent directions. Alignment was done by BWA (Burrows-Wheeler Aligner v0.7.13) “mem” algorithm using UCSC hg19 Human Reference Genome, variants and indels were called using GATK (Genome Analysis Toolkit v3.5) following the best practices, choosing HaplotypeCaller to generate a joint called Variant Call Format (VCF) file for all samples.
Genomic variant annotation was performed using ANNOVAR (ANNOtate VARiation v2016-02-01). A filter was applied to eliminate common variants as reported in the 1000 Genomes database. Data were visualized using the Integrative Genomics Viewer (IGV; Broad Institute, Cambridge, Mass.), and used to identify rare variants. To confirm accuracy of the sequencing read for rare variants, individual sample BAM files were visualized in the IGV software and analyzed for potential errors in sequencing. Using the 2017 release of the gnomAD browser (Broad Institute, Cambridge, Mass.), which contains exome sequence data from 123,136 individuals and whole genome sequencing from 15,496 individuals, remaining filtered variants were probed for previous reports in the literature. The Single Nucleotide Polymorphism database (dbSNP) was also referenced to determine whether rare variants identified by the gnomAD and 1000 Genomes databases were either novel or previously reported using this public-domain archive.
Statistical Methods
Throughout this paper, though figures present metabolic activity and cellular proliferation data as percentage of vehicle-treated control, statistical analyses were performed on raw data, following good statistical practice in pharmacology53. Specifically, in
To identify FDA-approved drugs with potential for repositioning in VS, we conducted a computational screen using the open-source drug repositioning platform ksRepo, developed to screen expression profiles from any microarray or sequencing platform against any available database of gene-drug interactions3. ksRepo uses a modified Kolmogorov-Smimov statistic to compare a ranked list of differentially expressed genes (DEGs) characteristic of a given disease with transcriptional signatures associated with drugs known to interact with those genes, as publicly stored in the Comparative Toxicogenomics Database11. From that list of drugs, ksRepo selects for compounds with entries in DrugBank, a compendium of FDA-approved drugs. The output is a list of FDA-approved drugs hypothesized to modulate genes with aberrant expression patterns in patients with disease (
To provide robust input to ksRepo, we conducted the largest meta-analysis of primary human VS tissue to date, comprising genome-wide transcriptional microarray data from 80 tumors and 16 control nerves (
Independently of our computational repositioning analysis, when gene expression data from our meta-analysis was input to Ingenuity Pathway Analysis (Qiagen), mifepristone was predicted as a significant upstream regulator of the resulting networks (p=4.26*10−5) and theorized to act upstream of inflammatory markers characteristic of VS, such as TNF and NFkB30,31 (
Administration of mifepristone to HEI-193 immortalized human schwannoma cells in culture produces a significant, dose-dependent response in metabolic activity (
Current, large-scale meta-analyses of drug toxicology, bioavailability, and efficacy in animal models reveal a shocking lack of predictive power when compared to human data35,36. Accordingly, the U.S. National Research Council has recommended the substitution of model animal testing with in vitro human cell-based assays and in silico modeling of diseases and networks37. We evaluated the effect of mifepristone applied directly to primary human VS cells. Fresh VS tissue samples from eight human patients undergoing tumor resection surgery were collected and schwannoma cells grown in the laboratory according to our published protocols38. Single-gene sequencing (Illumina MiSeq) of six treated VSs (
In a long-term clinical trial of mifepristone for unresectable meningioma, minor responses resulting in clinical benefit were noted in eight of 28 patients13, though a subsequent double-blind, randomized Phase III trial of 164 patients reported no difference between treatment and placebo39. To evaluate the effect of this drug on schwannoma cells in comparison to meningioma cells, we compared mifepristone-treated primary human VS and HEI-193 cells to immortalized human arachnoid cells in which the NF2 gene has been excised by CRISPR40. Primary VS cells responded more dramatically to mifepristone than human arachnoid cells with or without the NF2 gene (
The in silico repositioning of mifepristone for human VS using pooled human transcriptomic data, as well as the preclinical validation of this drug on primary human cells, constitutes a powerful case for the computational identification of novel indications for FDA-approved drugs. Mifepristone is safe and approved for human use and deserving of further attention for repurposing in a debilitating disease with no FDA-approved drug therapy.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 62/511,116, filed on May 25, 2017. The entire contents of the foregoing are hereby incorporated by reference.
This invention was made with Government support under Grant Nos. DC015824 and DC00038 awarded by the National Institutes of Health, and Grant No. W81XWH-15-1-0472 awarded by the United States Department of Defense. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/034168 | 5/23/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62511116 | May 2017 | US |
