Single Fraction and Hypofractionated Radiation Cause Cochlear Damage, Hearing Loss, and Reduced Viability of Merlin-Deficient Schwann Cells.

BACKGROUND: Vestibular schwannomas (VS) are benign intracranial tumors caused by loss of function of the merlin tumor suppressor. We tested three hypotheses related to radiation, hearing loss (HL), and VS cell survival: (1) radiation causes HL by injuring auditory hair cells (AHC), (2) fractionation reduces radiation-induced HL, and (3) single fraction and equivalent appropriately dosed multi-fractions are equally effective at controlling VS growth. We investigated the effects of single fraction and hypofractionated radiation on hearing thresholds in rats, cell death pathways in rat cochleae, and viability of human merlin-deficient Schwann cells (MD-SC). METHODS: Adult rats received cochlear irradiation with single fraction (0 to 18 Gray [Gy]) or hypofractionated radiation. Auditory brainstem response (ABR) testing was performed for 24 weeks. AHC viabilities were determined using immunohistochemistry. Neonatal rat cochleae were harvested after irradiation, and gene- and cell-based assays were conducted. MD-SCs were irradiated, and viability assays and immunofluorescence for DNA damage and cell cycle markers were performed. RESULTS: Radiation caused dose-dependent and progressive HL in rats and AHC losses by promoting expression of apoptosis-associated genes and proteins. When compared to 12 Gy single fraction, hypofractionation caused smaller ABR threshold and pure tone average shifts and was more effective at reducing MD-SC viability. CONCLUSIONS: Investigations into the mechanisms of radiation ototoxicity and VS radiobiology will help determine optimal radiation regimens and identify potential therapies to mitigate radiation-induced HL and improve VS tumor control.

Updates on Tumor Biology in Vestibular Schwannoma.

Vestibular schwannomas (VSs) are benign tumors that develop after biallelic inactivation of the neurofibromatosis type 2 (NF2) gene that encodes the tumor suppressor merlin. Merlin inactivation leads to cell proliferation by dysregulation of receptor tyrosine kinase signaling and other intracellular pathways. In VS without NF2 mutations, dysregulation of non-NF2 genes can promote pathways favoring cell proliferation and tumorigenesis. The tumor microenvironment of VS consists of multiple cell types that influence VS tumor biology through complex intercellular networking and communications.

RAD51 Inhibitor and Radiation Toxicity in Vestibular Schwannoma.

OBJECTIVE: To describe the RAD51 response (DNA repair) to radiation-induced DNA damage in patient-derived vestibular schwannoma (VS) cells and investigate the utility of RAD51 inhibitor (RI-1) in enhancing radiation toxicity. STUDY DESIGN: Basic and translational science. SETTING: Tertiary academic facility. METHODS: VS tumors (n = 10) were cultured on 96-well plates and 16-well slides, exposed to radiation (0, 6, 12, or 18 Gy), and treated with RI-1 (0, 5, or 10 µM). Immunofluorescence was performed at 6 hours for γ-H2AX (DNA damage marker), RAD51 (DNA repair protein), and p21 (cell cycle arrest protein). Viability assays were performed at 96 hours, and capillary Western blotting was utilized to determine RAD51 expression in naïve VS tumors (n = 5). RESULTS: VS tumors expressed RAD51. In cultured VS cells, radiation initiated dose-dependent increases in γ-H2AX and p21 expression. VS cells upregulated RAD51 to repair DNA damage following radiation. Addition of RI-1 reduced RAD51 expression in a dose-dependent manner and was associated with increased γ-H2AX levels and decreased viability in a majority of cultured VS tumors. CONCLUSION: VS may evade radiation injury by entering cell cycle arrest and upregulating RAD51-dependent repair of radiation-induced double-stranded breaks in DNA. Although there was variability in responses among individual primary VS cells, RAD51 inhibition with RI-1 reduced RAD51-dependent DNA repair to enhance radiation toxicity in VS cells. Further investigations are warranted to understand the mechanisms of radiation resistance in VS and determine whether RI-1 is an effective radiosensitizer in patients with VS.

Understanding the Radiobiology of Vestibular Schwannomas to Overcome Radiation Resistance.

Vestibular schwannomas (VS) are benign tumors arising from cranial nerve VIII that account for 8-10% of all intracranial tumors and are the most common tumors of the cerebellopontine angle. These tumors are typically managed with observation, radiation therapy, or microsurgical resection. Of the VS that are irradiated, there is a subset of tumors that are radioresistant and continue to grow; the mechanisms behind this phenomenon are not fully understood. In this review, the authors summarize how radiation causes cellular and DNA injury that can activate (1) checkpoints in the cell cycle to initiate cell cycle arrest and DNA repair and (2) key events that lead to cell death. In addition, we discuss the current knowledge of VS radiobiology and how it may contribute to clinical outcomes. A better understanding of VS radiobiology can help optimize existing treatment protocols and lead to new therapies to overcome radioresistance.

Characterization of UMi031-A-2 inducible pluripotent stem cell line with a neurofibromatosis type 2-associated mutation.

The UMi031-A-2 hiPSC line contains a CRISPR-induced homozygous, Neurofibromatosis Type 2 (NF2) mutation (L64P (CTG > CCG)) in the NF2 gene that encodes a merlin tumor suppressor. This line was generated from an unaffected iPSC line using CRISPR technology and characterized for pluripotency and karyotypic stability. The c.191 T > C variant in NF2 is associated with a syndromic nervous system tumor disorder leading to the development of bilateral vestibular schwannomas. Once differentiated into Schwann cells, UMi031-A-2 can serve as a resource for the analysis of signaling pathways deregulated upon merlin defects and provide a pre-clinical platform for testing therapies for NF2 schwannomas.

Stem Cells and Gene Therapy in Progressive Hearing Loss: the State of the Art.

Progressive non-syndromic sensorineural hearing loss (PNSHL) is the most common cause of sensory impairment, affecting more than a third of individuals over the age of 65. PNSHL includes noise-induced hearing loss (NIHL) and inherited forms of deafness, among which is delayed-onset autosomal dominant hearing loss (AD PNSHL). PNSHL is a prime candidate for genetic therapies due to the fact that PNSHL has been studied extensively, and there is a potentially wide window between identification of the disorder and the onset of hearing loss. Several gene therapy strategies exist that show potential for targeting PNSHL, including viral and non-viral approaches, and gene editing versus gene-modulating approaches. To fully explore the potential of these therapy strategies, a faithful in vitro model of the human inner ear is needed. Such models may come from induced pluripotent stem cells (iPSCs). The development of new treatment modalities by combining iPSC modeling with novel and innovative gene therapy approaches will pave the way for future applications leading to improved quality of life for many affected individuals and their families.

Euclidean Relationship between the Superior Semicircular Canal and the Arcuate Eminence.

Objective This study was aimed to better characterize the surgical anatomy of the floor of the middle cranial fossa using three dimensional Euclidean relationships between the arcuate eminence (AE), the superior semicircular canal (SSC), and the geniculate ganglion (GG). Study Design Submillimeter distances were recorded from computed tomography (CT) scans of 50 patients (100 sides). The AE, apex of the SSC, and the GG were identified and three dimensional distances measured. Setting The study was conducted at a tertiary academic teaching hospital. Main Outcome Measures In this study, Euclidean distance was obtained from AE to SSC by using a fixed anatomical landmark (GG) as the origin. Results On average, the AE is 2.1 ± 0.3 mm lateral, 2.5 ± 0.1 mm superior, and 2.1 ± 0.3 posterior to the SSC. Thirty percent (30/100) of patients had an AE that was less than 2 mm superior to SSC. The AE was medial to the SCC in 13% samples and anterior to the SSC in 18% samples. The results also show that there was no difference in mean distance between sides (1.08 mm; 95% confidence interval [CI] = - 2.67-0.52; p -value = 0.29) or gender (0.56 mm; 95% CI = - 1.34, 2.45; p -value = 0.86). Conclusions This study represents a comprehensive analysis of the relational anatomy of the floor of the middle fossa to date. In quantifying relationships between the AE, SSC, and GG, and by understanding the variability of these relationships in some planes, the middle fossa surgeon can feel more comfortable with this most challenging approach.

GRP78/Dna K Is a Target for Nexavar/Stivarga/Votrient in the Treatment of Human Malignancies, Viral Infections and Bacterial Diseases.

Prior tumor cell studies have shown that the drugs sorafenib (Nexavar) and regorafenib (Stivarga) reduce expression of the chaperone GRP78. Sorafenib/regorafenib and the multi-kinase inhibitor pazopanib (Votrient) interacted with sildenafil (Viagra) to further rapidly reduce GRP78 levels in eukaryotes and as single agents to reduce Dna K levels in prokaryotes. Similar data were obtained in tumor cells in vitro and in drug-treated mice for: HSP70, mitochondrial HSP70, HSP60, HSP56, HSP40, HSP10, and cyclophilin A. Prolonged 'rafenib/sildenafil treatment killed tumor cells and also rapidly decreased the expression of: the drug efflux pumps ABCB1 and ABCG2; and NPC1 and NTCP, receptors for Ebola/Hepatitis A and B viruses, respectively. Pre-treatment with the 'Rafenib/sildenafil combination reduced expression of the Coxsackie and Adenovirus receptor in parallel with it also reducing the ability of a serotype 5 Adenovirus or Coxsackie virus B4 to infect and to reproduce. Sorafenib/pazopanib and sildenafil was much more potent than sorafenib/pazopanib as single agents at preventing Adenovirus, Mumps, Chikungunya, Dengue, Rabies, West Nile, Yellow Fever, and Enterovirus 71 infection and reproduction. 'Rafenib drugs/pazopanib as single agents killed laboratory generated antibiotic resistant E. coli which was associated with reduced Dna K and Rec A expression. Marginally toxic doses of 'Rafenib drugs/pazopanib restored antibiotic sensitivity in pan-antibiotic resistant bacteria including multiple strains of blakpc Klebsiella pneumoniae. Thus, Dna K is an antibiotic target for sorafenib, and inhibition of GRP78/Dna K has therapeutic utility for cancer and for bacterial and viral infections.

OSU-03012 and Viagra Treatment Inhibits the Activity of Multiple Chaperone Proteins and Disrupts the Blood-Brain Barrier: Implications for Anti-Cancer Therapies.

We examined the interaction between OSU-03012 (also called AR-12) with phosphodiesterase 5 (PDE5) inhibitors to determine the role of the chaperone glucose-regulated protein (GRP78)/BiP/HSPA5 in the cellular response. Sildenafil (Viagra) interacted in a greater than additive fashion with OSU-03012 to kill stem-like GBM cells. Treatment of cells with OSU-03012/sildenafil: abolished the expression of multiple oncogenic growth factor receptors and plasma membrane drug efflux pumps and caused a rapid degradation of GRP78 and other HSP70 and HSP90 family chaperone proteins. Decreased expression of plasma membrane receptors and drug efflux pumps was dependent upon enhanced PERK-eIF2α-ATF4-CHOP signaling and was blocked by GRP78 over-expression. In vivo OSU-03012/sildenafil was more efficacious than treatment with celecoxib and sildenafil at killing tumor cells without damaging normal tissues and in parallel reduced expression of ABCB1 and ABCG2 in the normal brain. The combination of OSU-03012/sildenafil synergized with low concentrations of sorafenib to kill tumor cells, and with lapatinib to kill ERBB1 over-expressing tumor cells. In multiplex assays on plasma and human tumor tissue from an OSU-03012/sildenafil treated mouse, we noted a profound reduction in uPA signaling and identified FGF and JAK1/2 as response biomarkers for potentially suppressing the killing response. Inhibition of FGFR signaling and to a lesser extent JAK1/2 signaling profoundly enhanced OSU-03012/sildenafil lethality.