Targeting histone deacetylase 6 (HDAC6) to enhance radiation therapy in meningiomas in a 2D and 3D in vitro study.

BACKGROUND: External radiation therapy (RT) is often a primary treatment for inoperable meningiomas in the absence of established chemotherapy. Histone deacetylase 6 (HDAC6) overexpression, commonly found in cancer, is acknowledged as a driver of cellular growth, and inhibiting HDACs holds promise in improving radiotherapeutic efficacy. Downregulation of HDAC6 facilitates the degradation of ß-catenin. This protein is a key element in the Wnt/ß-catenin signalling pathway, contributing to the progression of meningiomas. METHODS: In order to elucidate the associations and therapeutic potential of HDAC6 inhibitors (HDAC6i) in conjunction with RT, we administered Cay10603, HDAC6i, to both immortalised and patient-derived meningioma cells prior to RT in this study. FINDINGS: Our findings reveal an increase in HDAC6 expression following exposure to RT, which is effectively mitigated with pre-treated Cay10603. The combination of Cay10603 with RT resulted in a synergistic augmentation of cytotoxic effects, as demonstrated through a range of functional assays conducted in both 2D as well as 3D settings; the latter containing syngeneic tumour microenvironment (TME). Radiation-induced DNA damage was augmented by pre-treatment with Cay10603, concomitant with the inhibition of ß-catenin and minichromosome maintenance complex component 2 (MCM2) accumulation within the nucleus. This subsequently inhibited c-myc oncogene expression. INTERPRETATION: Our findings demonstrate the therapeutic potential of Cay10603 to improve the radiosensitisation and provide rationale for combining HDAC6i with RT for the treatment of meningioma. FUNDING: This work was funded by Brain Tumour Research Centre of Excellence award to C Oliver Hanemann.

A novel patient-derived meningioma spheroid model as a tool to study and treat epithelial-to-mesenchymal transition (EMT) in meningiomas.

Meningiomas are the most common intracranial brain tumours. These tumours are heterogeneous and encompass a wide spectrum of clinical aggressivity. Treatment options are limited to surgery and radiotherapy and have a risk of post-operative morbidities and radiation neurotoxicity, reflecting the need for new therapies. Three-dimensional (3D) patient-derived cell culture models have been shown to closely recapitulate in vivo tumour biology, including microenvironmental interactions and have emerged as a robust tool for drug development. Here, we established a novel easy-to-use 3D patient-derived meningioma spheroid model using a scaffold-free approach. Patient-derived meningioma spheroids were characterised and compared to patient tissues and traditional monolayer cultures by histology, genomics, and transcriptomics studies. Patient-derived meningioma spheroids closely recapitulated morphological and molecular features of matched patient tissues, including patient histology, genomic alterations, and components of the immune microenvironment, such as a CD68 + and CD163 + positive macrophage cell population. Comprehensive transcriptomic profiling revealed an increase in epithelial-to-mesenchymal transition (EMT) in meningioma spheroids compared to traditional monolayer cultures, confirming this model as a tool to elucidate EMT in meningioma. Therefore, as proof of concept study, we developed a treatment strategy to target EMT in meningioma. We found that combination therapy using the MER tyrosine kinase (MERTK) inhibitor UNC2025 and the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) effectively decreased meningioma spheroid viability and proliferation. Furthermore, we demonstrated this combination therapy significantly increased the expression of the epithelial marker E-cadherin and had a repressive effect on WHO grade 2-derived spheroid invasion, which is suggestive of a partial reversal of EMT in meningioma spheroids.

Overexpression of Both Human Sodium Iodide Symporter (NIS) and BRG1-Bromodomain Synergistically Enhances Radioiodine Sensitivity by Stabilizing p53 through NPM1 Expression.

Improved therapeutic strategies are required to minimize side effects associated with radioiodine gene therapy to avoid unnecessary damage to normal cells and radiation-induced secondary malignancies. We previously reported that codon-optimized sodium iodide symporter (oNIS) enhances absorption of I-131 and that the brahma-associated gene 1 bromodomain (BRG1-BRD) causes inefficient DNA damage repair after high-energy X-ray therapy. To increase the therapeutic effect without applying excessive radiation, we considered the combination of oNIS and BRG1-BRD as gene therapy for the most effective radioiodine treatment. The antitumor effect of I-131 with oNIS or oNIS+BRD expression was examined by tumor xenograft models along with functional assays at the cellular level. The synergistic effect of both BRG1-BRD and oNIS gene overexpression resulted in more DNA double-strand breaks and led to reduced cell proliferation/survival rates after I-131 treatment, which was mediated by the p53/p21 pathway. We found increased p53, p21, and nucleophosmin 1 (NPM1) in oNIS- and BRD-expressing cells following I-131 treatment, even though the remaining levels of citrulline and protein arginine deiminase 4 (PAD4) were unchanged at the protein level.

SPRTN protease-cleaved MRE11 decreases DNA repair and radiosensitises cancer cells.

The human MRE11/RAD50/NBS1 (MRN) complex plays a crucial role in sensing and repairing DNA DSB. MRE11 possesses dual 3'-5' exonuclease and endonuclease activity and forms the core of the multifunctional MRN complex. We previously identified a C-terminally truncated form of MRE11 (TR-MRE11) associated with post-translational MRE11 degradation. Here we identified SPRTN as the essential protease for the formation of TR-MRE11 and characterised the role of this MRE11 form in its DNA damage response (DDR). Using tandem mass spectrometry and site-directed mutagenesis, the SPRTN-dependent cleavage site for MRE11 was identified between 559 and 580 amino acids. Despite the intact interaction of TR-MRE11 with its constitutive core complex proteins RAD50 and NBS1, both nuclease activities of truncated MRE11 were dramatically reduced due to its deficient binding to DNA. Furthermore, lack of the MRE11 C-terminal decreased HR repair efficiency, very likely due to abolished recruitment of TR-MRE11 to the sites of DNA damage, which consequently led to increased cellular radiosensitivity. The presence of this DNA repair-defective TR-MRE11 could explain our previous finding that the high MRE11 protein expression by immunohistochemistry correlates with improved survival following radical radiotherapy in bladder cancer patients.

[18F]CB251 PET/MR imaging probe targeting translocator protein (TSPO) independent of its Polymorphism in a Neuroinflammation Model.

The 18 kDa translocator protein (TSPO) has been proposed as a biomarker for the detection of neuroinflammation. Although various PET probes targeting TSPO have been developed, a highly selective probe for detecting TSPO is still needed because single nucleotide polymorphisms in the human TSPO gene greatly affect the binding affinity of TSPO ligands. Here, we describe the visualization of neuroinflammation with a multimodality imaging system using our recently developed TSPO-targeting radionuclide PET probe [18F]CB251, which is less affected by TSPO polymorphisms. Methods: To test the selectivity of [18F]CB251 for TSPO polymorphisms, 293FT cells expressing polymorphic TSPO were generated by introducing the coding sequences of wild-type (WT) and mutant (Alanine → Threonine at 147th Amino Acid; A147T) forms. Competitive inhibition assay was conducted with [3H]PK11195 and various TSPO ligands using membrane proteins isolated from 293FT cells expressing TSPO WT or mutant-A147T, representing high-affinity binder (HAB) or low-affinity binder (LAB), respectively. IC50 values of each ligand to [3H]PK11195 in HAB or LAB were measured and the ratio of IC50 values of each ligand to [3H]PK11195 in HAB to LAB was calculated, indicating the sensitivity of TSPO polymorphism. Cellular uptake of [18F]CB251 was measured with different TSPO polymorphisms, and phantom studies of [18F]CB251-PET using 293FT cells were performed. To test TSPO-specific cellular uptake of [18F]CB251, TSPO expression was regulated with pCMV-TSPO (or shTSPO)/eGFP vector. Intracranial lipopolysaccharide (LPS) treatment was used to induce regional inflammation in the mouse brain. Gadolinium (Gd)-DOTA MRI was used to monitor the disruption of the blood-brain barrier (BBB) and infiltration by immune cells. Infiltration of peripheral immune cells across the BBB, which exacerbates neuroinflammation to produce higher levels of neurotoxicity, was also monitored with bioluminescence imaging (BLI). Peripheral immune cells isolated from luciferase-expressing transgenic mice were transferred to syngeneic inflamed mice. Neuroinflammation was monitored with [18F]CB251-PET/MR and BLI. To evaluate the effects of anti-inflammatory agents on intracranial inflammation, an inflammatory cytokine inhibitor, 2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid methyl ester (CDDO-Me) was administered in intracranial LPS challenged mice. Results: The ratio of IC50 values of [18F]CB251 in HAB to LAB indicated similar binding affinity to WT and mutant TSPO and was less affected by TSPO polymorphisms. [18F]CB251 was specific for TSPO, and its cellular uptake reflected the amount of TSPO. Higher [18F]CB251 uptake was also observed in activated immune cells. Simultaneous [18F]CB251-PET/MRI showed that [18F]CB251 radioactivity was co-registered with the MR signals in the same region of the brain of LPS-injected mice. Luciferase-expressing peripheral immune cells were located at the site of LPS-injected right striatum. Quantitative evaluation of the anti-inflammatory effect of CDDO-Me on neuroinflammation was successfully monitored with TSPO-targeting [18F]CB251-PET/MR and BLI. Conclusion: Our results indicate that [18F]CB251-PET has great potential for detecting neuroinflammation with higher TSPO selectivity regardless of polymorphisms. Our multimodal imaging system, [18F]CB251-PET/MRI, tested for evaluating the efficacy of anti-inflammatory agents in preclinical studies, might be an effective method to assess the severity and therapeutic response of neuroinflammation.

New Drug Approvals in 2018 - Another Record Year!

In 2018, the US Food and Drug Administration approved 59 novel drugs. This all-time record was due primarily to the expedited review pathways; 43 of the 59 (73%) novel drug approvals were designated in an expedited review pathway, and 34 of the 59 (58%) were approved for treatment of rare diseases. A review of these novel drugs is summarized.

Hexokinase-II Inhibition Synergistically Augments the Anti-tumor Efficacy of Sorafenib in Hepatocellular Carcinoma.

This study aimed to examine whether inhibition of hexokinase (HK)-II activity enhances the efficacy of sorafenib in in-vivo models of hepatocellular carcinoma (HCC), and to evaluate the prognostic implication of HK-II expression in patients with HCC. We used 3-bromopyruvate (3-BP), a HK-II inhibitor to target HK-II. The human HCC cell line was tested as both subcutaneous and orthotopic tumor xenograft models in BALB/c nu/nu mice. The prognostic role of HK-II was evaluated in data from HCC patients in The Cancer Genome Atlas (TCGA) database and validated in patients treated with sorafenib. Quantitative real-time PCR, western blot analysis, and immunohistochemical staining revealed that HK-II expression is upregulated in the presence of sorafenib. Further analysis of the endoplasmic reticulum-stress network model in two different murine HCC models showed that the introduction of additional stress by 3-BP treatment synergistically increased the in vivo/vitro efficacy of sorafenib. We found that HCC patients with increased HK-II expression in the TCGA database showed poor overall survival, and also confirmed similar results for TCGA database HCC patients who had undergone sorafenib treatment. These results suggest that HK-II is a promising therapeutic target to enhance the efficacy of sorafenib and that HK-II expression might be a prognostic factor in HCC.

MRE11A Isoform Expression Associated with Outcome Following Radiotherapy in Muscle-Invasive Bladder Cancer does not Alter Cell Survival and DNA Double-Strand Break Repair Following Ionising Radiation.

BACKGROUND: DNA double strand breaks are the cytotoxic lesions produced by ionising radiation. Critical for the repair of these lesions is the DNA damage response protein MRE11 which, in a complex with RAD50 and NBS1, mediates DNA damage signalling and double-strand break repair. We previously found the presence of an MRE11 germline single nucleotide polymorphism (SNP), rs1805363 (G > A), to be associated with poor outcome following radiotherapy (RT) and increased expression of MRE11 isoform 2 in a limited panel of bladder cancer cell lines and tumours. OBJECTIVES: To look for further evidence in support of the SNP/isoform association in a larger panel of germline and tumour samples donated by patients diagnosed with invasive bladder cancer, and to test the hypothesis that bladder cancer cells expressing MRE11 isoform 2 would be more radio resistant than cells expressing MRE11 isoform 1. METHODS: Germline DNA from 189 patients with invasive bladder cancer (141 T2, 48 T1) was genotyped for the rs1805363 G > A SNP. Loss of heterozygosity was determined by genotyping tumour DNA in 17GA germline patients. The Cancer Genome Atlas was mined to correlate presence of the GA germline genotype with MRE11 isoform expression. We used colony formation assays and γH2AX foci kinetics after ionising radiation in RT112 MRE11 knockdown cells expressing ectopic MRE11 isoform 1 or 2. RESULTS: Of the 189 germline DNA samples, 22 contained both the A minor allele and G major allele with the remaining wild type containing only the G major allele. LOH was identified in seven of 17 available tumour samples. Tumour MRE11 isoform 2 expression was found to be significantly higher (p = 0.007) in patients's samples containing the A minor allele compared to those with only the G major allele (n = 23). In the TCGA database we found 16% (66 out of 406) of bladder tumours heterozygous for the SNP and only two homozygous, and a significant relative increase of isoform 2 usage (p = 0.017). We identified no significant difference in radio sensitivity between bladder cancer cells expressing either MRE11 isoform. CONCLUSIONS: In this study the MRE11 isoform 2 was not found to be associated with increased cellular sensitivity to radiation. We conclude that the previously reported association between the germline rs1805363 SNP and poor survival in MIBC patients following RT is unlikely to be related to the DNA damage response function of MRE11 isoform 2.

Effect of perioperative oral nutritional supplementation in malnourished patients who undergo gastrectomy: A prospective randomized trial.

BACKGROUND: The aim of this study was to examine the effect of a perioperative oral nutritional supplement in malnourished patients who undergo gastrectomy. METHODS: Patients who were determined as being moderately or severely malnourished according to a patient-generated subjective global assessment or who had a body mass index <18.5, were enrolled. The oral nutritional supplement group received 500 mL/d of standard oral nutritional supplement for 2 weeks before gastrectomy and for 4 weeks postoperatively. The primary endpoint was postoperative complications (Clavien-Dindo classification ≥II). The secondary endpoints included body weight changes, biochemical parameters, and quality of life survey results. RESULTS: A total of 127 patients (65 in the oral nutritional supplement group and 62 in the control group) were enrolled. The complication rates were not significantly different (29.2% versus 37.1%, P = .346). However, the incidences of overall complications, complications persisting until postoperative week 3 or 5, and severe complications (grade ≥IIIa) were significantly lower in the oral nutritional supplement group for patients with patient-generated subjective global assessment grade C. Total lymphocyte counts were significantly higher in the oral nutritional supplement group at postoperative weeks 3 and 5. For most patients, oral nutritional supplement was well tolerated preoperatively. However, only 26.2% and 50.8% of the patients in the oral nutritional supplement group could consume >250 mL/d of oral nutritional supplement postoperatively during the 2nd and 4th weeks, respectively. CONCLUSIONS: The routine application of perioperative oral nutritional supplement is not recommended for malnourished patients receiving gastrectomy. However, perioperative standard oral nutritional supplement administration may reduce the incidence, severity, and duration of complications after gastrectomy in severely malnourished patients (patient-generated subjective global assessment grade C).

Thyroid-Related Protein Expression in the Human Thymus.

Radioiodine whole body scan (WBS), related to sodium iodide symporter (NIS) function, is widely used to detect recurrence/metastasis in postoperative patients with thyroid cancer. However, the normal thymic uptake of radioiodine has occasionally been observed in young patients. We evaluated the expression of thyroid-related genes and proteins in the human thymus. Thymic tissues were obtained from 22 patients with thyroid cancer patients of all ages. The expression of NIS, thyroid-stimulating hormone receptor (TSHR), thyroperoxidase (TPO), and thyroglobulin (Tg) was investigated using immunohistochemistry and quantitative RT-PCR. NIS and TSHR were expressed in 18 (81.8%) and 19 samples (86.4%), respectively, whereas TPO was expressed in five samples (22.7%). Three thyroid-related proteins were localized to Hassall's corpuscles and thymocytes. In contrast, Tg was detected in a single patient (4.5%) localized to vascular endothelial cells. The expression of thyroid-related proteins was not increased in young thymic tissues compared to that in old thymic tissues. In conclusion, the expression of NIS and TSHR was detected in the majority of normal thymus samples, whereas that of TPO was detected less frequently, and that of Tg was detected rarely. The increased thymic uptake of radioiodine in young patients is not due to the increased expression of NIS.