The importance of routine genetic testing in pediatric epilepsy surgery.

Genetic variants in relevant genes coexisting with MRI lesions in children with drug-resistant epilepsy (DRE) can negatively influence epilepsy surgery outcomes. Still, presurgical evaluation does not include genetic diagnostics routinely. Here, we report our presurgical evaluation algorithm that includes routine genetic testing. We analyzed retrospectively the data of 68 children with DRE operated at a mean age of 7.8 years (IQR: 8.1 years) at our center. In 49 children, genetic test results were available. We identified 21 gene variants (ACMG III: n = 7, ACMG IV: n = 2, ACMG V: n = 12) in 19 patients (45.2%) in the genes TSC1, TSC2, MECP2, DEPDC5, HUWE1, GRIN1, ASH1I, TRIO, KIF5C, CDON, ANKD11, TGFBR2, ATN1, COL4A1, JAK2, KCNQ2, ATP1A2, and GLI3 by whole-exome sequencing as well as deletions and duplications by array CGH in six patients. While the results did not change the surgery indication, they supported counseling with respect to postoperative chance of seizure freedom and weaning of antiseizure medication (ASM). The presence of genetic findings leads to the postoperative retention of at least one ASM. In our cohort, the International League against Epilepsy (ILAE) seizure outcome did not differ between patients with and without abnormal genetic findings. However, in the 7/68 patients with an unsatisfactory ILAE seizure outcome IV or V 12 months postsurgery, 2 had an abnormal or suspicious genetic finding as a putative explanation for persisting seizures postsurgery, and 3 had received palliative surgery including one TSC patient. This study highlights the importance of genetic testing in children with DRE to address putative underlying germline variants as genetic epilepsy causes or predisposing factors that guide patient and/or parent counseling on a case-by-case with respect to their individual chance of postoperative seizure freedom and ASM weaning. PLAIN LANGUAGE SUMMARY: Genetic variants in children with drug-resistant epilepsy (DRE) can negatively influence epilepsy surgery outcomes. However, presurgical evaluation does not include genetic diagnostics routinely. This retrospective study analyzed the genetic testing results of the 68 pediatric patients who received epilepsy surgery in our center. We identified 21 gene variants by whole-exome sequencing as well as deletions and duplications by array CGH in 6 patients. These results highlight the importance of genetic testing in children with DRE to guide patient and/or parent counseling on a case-by-case with respect to their individual chance of postoperative seizure freedom and ASM weaning.

Efficacy of Immediate Antihypertensive Treatment in Patients With Acute Ischemic Stroke With Different Blood Pressure Genetic Variants.

BACKGROUND: It remains unclear whether blood pressure (BP) genetic variants could modify the efficacy of immediate antihypertensive treatment after acute ischemic stroke. We conducted a secondary analysis of the CATIS (China Antihypertensive Trial in Acute Ischemic Stroke) to investigate the effect of early antihypertensive treatment on clinical outcomes among patients with acute ischemic stroke according to 5 BP-associated genetic variants. METHODS: The CATIS randomized 4071 patients with acute ischemic stroke with elevated systolic BP to receive antihypertensive treatment or discontinue all antihypertensive agents during hospitalization. Randomization was conducted centrally and was stratified by participating hospitals and use of antihypertensive medications. Five BP-associated single nucleotide polymorphisms (rs16849225, rs17030613, rs1173766, rs6825911, and rs35444 in FIGN-GRB14, ST7L-CAPZA1, NPR3, ENPEP, and near TBX3, respectively) were genotyped among 2590 patients. The primary outcome was a combination of death and major disability at 14 days or hospital discharge. A weighted BP genetic risk score was constructed by the 5 single nucleotide polymorphisms. RESULTS: At 14 days or hospital discharge, the primary outcome was not significantly different between antihypertensive treatment and control groups based on genotype subgroups for all 5 single nucleotide polymorphisms (all P>0.05 for interaction). In addition, the BP genetic risk score did not modify the effect of antihypertensive treatment. The odds ratios (95% CIs) for the primary outcome were 0.95 (0.71-1.26), 1.08 (0.80-1.44), and 0.91 (0.69-1.22) in patients with low, intermediate, and high BP genetic risk score, respectively (P=0.88 for interaction). CONCLUSIONS: Early antihypertensive treatment had a neutral effect on clinical outcomes among patients with acute ischemic stroke according to 5 BP-associated genetic variants. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT01840072.

Inhibition of CK2/ING4 Pathway Facilitates Non-Small Cell Lung Cancer Immunotherapy.

Immune cells can protect against tumor progression by killing cancer cells, while aberrant expression of the immune checkpoint protein PD-L1 (programmed death ligand 1) in cancer cells facilitates tumor immune escape and inhibits anti-tumor immunotherapy. As a serine/threonine kinase, CK2 (casein kinase 2) regulates tumor progression by multiple pathways, while it is still unclear the effect of CK2 on tumor immune escape. Here it is found that ING4 induced PD-L1 autophagic degradation and inhibites non-small cell lung cancer (NSCLC) immune escape by increasing T cell activity. However, clinical analysis suggests that high expression of CK2 correlates with low ING4 protein level in NSCLC. Further analysis shows that CK2 induce ING4-S150 phosphorylation leading to ING4 ubiquitination and degradation by JFK ubiquitin ligase. In contrast, CK2 gene knockout increases ING4 protein stability and T cell activity, subsequently, inhibites NSCLC immune escape. Furthermore, the combined CK2 inhibitor with PD-1 antibody effectively enhances antitumor immunotherapy. These findings provide a novel strategy for cancer immunotherapy.

MGMT promoter methylation status prediction using MRI scans? An extensive experimental evaluation of deep learning models.

The number of studies on deep learning for medical diagnosis is expanding, and these systems are often claimed to outperform clinicians. However, only a few systems have shown medical efficacy. From this perspective, we examine a wide range of deep learning algorithms for the assessment of glioblastoma - a common brain tumor in older adults that is lethal. Surgery, chemotherapy, and radiation are the standard treatments for glioblastoma patients. The methylation status of the MGMT promoter, a specific genetic sequence found in the tumor, affects chemotherapy's effectiveness. MGMT promoter methylation improves chemotherapy response and survival in several cancers. MGMT promoter methylation is determined by a tumor tissue biopsy, which is then genetically tested. This lengthy and invasive procedure increases the risk of infection and other complications. Thus, researchers have used deep learning models to examine the tumor from brain MRI scans to determine the MGMT promoter's methylation state. We employ deep learning models and one of the largest public MRI datasets of 585 participants to predict the methylation status of the MGMT promoter in glioblastoma tumors using MRI scans. We test these models using Grad-CAM, occlusion sensitivity, feature visualizations, and training loss landscapes. Our results show no correlation between these two, indicating that external cohort data should be used to verify these models' performance to assure the accuracy and reliability of deep learning systems in cancer diagnosis.

Gene mutation profiling and clinical significances in patients with renal cell carcinoma.

OBJECTIVES: The pathological mechanisms of patients with Renal Cell Carcinoma (RCC) remain defined. This study aimed to evaluate relationships between the landscape of gene mutations and their clinical significance in RCC patients. METHODS: Tissue and peripheral blood samples of 42 patients with RCC were collected and performed for the Next Generation Sequencing (NGS) with Geneseeq PrimeTM 425-gene panel probes. Their landscapes of gene mutation were analyzed. We also carried out an evaluation of Tumor-Node-Metastasis (TNM) staging, RENAL nephelometry score, surgery, and targeted drug treatment of patients. Then we compared the correlations of landscape in gene mutations and the prognosis. RESULTS: The most common gene alternations, including BAP1, PBRM1, SETD2, CSF1R, NPM1, EGFR, POLE, RB1, and VHL genes, were identified in tissue and blood samples of 75% of patients. EGFR, POLE, and RB1 gene mutations frequently occurred in relapsed and metastatic patients. BAP1, CCND2, KRAS, PTPN11, ERBB2/3, JAK2, and POLE were presented in the patients with > 9 RENAL nephelometry score. Univariable analysis indicated that SETD2, BAP1, and PBRM1 genes were key factors for Disease-Free Survival (DFS). Multivariable analysis confirmed that mutated SETD1, NPM1, and CSF1R were critical factors for the Progression Free Survival (PFS) of RCC patients with target therapy. CONCLUSIONS: Wild-type PBRM1 and mutated BAP1 in patients with RCC were strongly associated with the outcomes of the patient. The PFS of the patients with SETD2, NPM1, and CSF1R mutations were significantly shorter than those patients without variants.

BAP1-related signature predicts benefits from immunotherapy over VEGFR/mTOR inhibitors in ccRCC: a retrospective analysis of JAVELIN Renal 101 and checkmate-009/010/025 trials.

BACKGROUND: In patients with advanced clear cell renal cell carcinoma, despite the undoubted benefits from immune checkpoint inhibitor (ICI)-based therapies over monotherapies of angiogenic/mTOR inhibitors in the intention-to-treat population, approximately a quarter of the patients can scarcely gain advantage from ICIs, prompting the search for predictive biomarkers for patient selection. METHODS: Clinical and multi-omic data of 2428 ccRCC patients were obtained from The Cancer Genome Atlas (TCGA, n = 537), JAVELIN Renal 101 (avelumab plus axitinib vs. sunitinib, n = 885), and CheckMate-009/010/025 (nivolumab vs. everolimus, n = 1006). RESULTS: BAP1 mutations were associated with large progression-free survival (PFS) benefits from ICI-based immunotherapies over sunitinib/everolimus (pooled estimate of interaction HR = 0.71, 95% CI 0.51-0.99, P = 0.045). Using the top 20 BAP1 mutation-associated differentially expressed genes (DEGs) generated from the TCGA cohort, we developed the BAP1-score, negatively correlated with angiogenesis and positively correlated with multiple immune-related signatures concerning immune cell infiltration, antigen presentation, B/T cell receptor, interleukin, programmed death-1, and interferon. A high BAP1-score indicated remarkable PFS benefits from ICI-based immunotherapies over angiogenic/mTOR inhibitors (avelumab plus axitinib vs. sunitinib: HR = 0.55, 95% CI 0.43-0.70, P < 0.001; nivolumab vs. everolimus: HR = 0.72, 95% CI 0.52-1.00, P = 0.045), while these benefits were negligible in the low BAP1-score subgroup (HR = 1.16 and 1.02, respectively). CONCLUSION: In advanced ccRCCs, the BAP1-score is a biologically and clinically significant predictor of immune microenvironment and the clinical benefits from ICI-based immunotherapies over angiogenic/mTOR inhibitors, demonstrating its potential utility in optimizing the personalized therapeutic strategies in patients with advanced ccRCC.

BAG1, MGMT, FOXO1, and DNAJA1 as potential drug targets for radiosensitizing cancer cell lines.

BACKGROUND AND PURPOSE: Activation of some signaling pathways can promote cell survival and have a negative impact on tumor response to radiotherapy. Here, the role of differences in expression levels of genes related to the poly(ADP-ribose) polymerase-1 (PARP-1), heat shock protein 90 (Hsp90), B-cell lymphoma 2 (Bcl-2), and phosphoinositide 3-kinase (PI3K) pathways in the survival or death of cells following X-ray exposure was investigated. METHODS: Eight human cell cultures (MCF-7 and MDA-MB-231: breast cancers; MCF-12A: apparently normal breast; A549: lung cancer; L132: normal lung; G28, G44 and G112: glial cancers) were irradiated with X-rays. The colony-forming and real-time PCR based on a custom human pathway RT2 Profiler PCR Array assays were used to evaluate cell survival and gene expression, respectively. RESULTS: The surviving fractions at 2 Gy for the cell lines, in order of increasing radioresistance, were found to be as follows: MCF-7 (0.200 ± 0.011), G44 (0.277 ± 0.065), L132 (0.367 ± 0.023), MDA-MB-231 (0.391 ± 0.057), G112 (0.397 ± 0.113), A549 (0.490 ± 0.048), MCF-12A (0.526 ± 0.004), and G28 (0.633 ± 0.094). The rank order of radioresistance at 6 Gy was: MCF-7 < L132 < G44 < MDA-MB-231 < A549 < G28 < G112 < MCF-12A. PCR array data analysis revealed that several genes were differentially expressed between irradiated and unirradiated cell cultures. The following genes, with fold changes: BCL2A1 (21.91), TP53 (8743.75), RAD51 (11.66), FOX1 (65.86), TCP1 (141.32), DNAJB1 (3283.64), RAD51 (51.52), and HSPE1 (12887.29) were highly overexpressed, and BAX (-127.21), FOX1 (-81.79), PDPK1 (-1241.78), BRCA1 (-8.70), MLH1 (-12143.95), BCL2 (-18.69), CCND1 (-46475.98), and GJA1 (-2832.70) were highly underexpressed in the MDA-MB-231, MCF-7, MCF-12A, A549, L132, G28, G44, and G112 cell lines, respectively. The radioresistance in the malignant A549 and G28 cells was linked to upregulation in the apoptotic, DNA repair, PI3K, and Hsp90 pathway genes BAG1, MGMT, FOXO1, and DNAJA1, respectively, and inhibition of these genes resulted in significant radiosensitization. CONCLUSIONS: Targeting BAG1, MGMT, FOXO1, and DNAJA1 with specific inhibitors might effectively sensitize radioresistant tumors to radiotherapy.

Drug delivery in glioblastoma therapy: a review on nanoparticles targeting MGMT-mediated resistance.

INTRODUCTION: Glioblastoma multiforme (GBM) is the deadliest type of brain cancer with poor response to the available therapies, mainly due to intrinsic resistance mechanisms. Chemotherapy is based on alkylating agents, but DNA-repair mechanisms can revert this cytotoxic effect. O6-methylguanine-DNA methyltransferase (MGMT) protein is the primary mechanism for GBM resistance. Therefore, different strategies to suppress its activity have been explored. However, their clinical use has been hindered due to the high toxicity of MGMT inhibitors verified in clinical trials. AREAS COVERED: This review article aims to provide the current progress in the development of novel drug delivery systems (DDS) to overcome this resistance. Here, we also review the current knowledge on MGMT-mediated resistance and the clinical outcomes and potential risks of using MGMT inhibitors. EXPERT OPINION: To overcome therapeutic limitations, nano-based approaches have been proposed as a suitable solution to improve drug accumulation in the brain tumor tissue and decrease systemic toxicity. DDS to overcome MGMT-mediated resistance in GBM have been mostly developed to deliver MGMT inhibitors and for gene therapy to modulate MGMT gene expression.

Btg2 mutation induces renal injury and impairs blood pressure control in female rats.

Hypertension (HTN) is a complex disease influenced by heritable genetic elements and environmental interactions. Dietary salt is among the most influential modifiable factors contributing to increased blood pressure (BP). It is well established that men and women develop BP impairment in different patterns and a recent emphasis has been placed on identifying mechanisms leading to the differences observed between the sexes in HTN development. The current work reported here builds on an extensive genetic mapping experiment that sought to identify genetic determinants of salt-sensitive (SS) HTN using the Dahl SS rat. BTG antiproliferation factor 2 (Btg2) was previously identified by our group as a candidate gene contributing to SS HTN in female rats. In the current study, Btg2 was mutated using transcription activator-like effector nuclease (TALEN)-targeted gene disruption on the SSBN congenic rat background. The Btg2 mutated rats exhibited impaired BP and proteinuria responses to a high-salt diet compared with wild-type rats. Differences in body weight, mutant pup viability, skeletal morphology, and adult nephron density suggest a potential role for Btg2 in developmental signaling pathways. Subsequent cell cycle gene expression assessment provides several additional signaling pathways that Btg2 may function through during salt handling in the kidney. The expression analysis also identified several potential upstream targets that can be explored to further isolate therapeutic approaches for SS HTN.

Phase I/II trial of meclofenamate in progressive MGMT-methylated glioblastoma under temozolomide second-line therapy-the MecMeth/NOA-24 trial.

BACKGROUND: Glioblastoma is the most frequent and malignant primary brain tumor. Even in the subgroup with O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation and favorable response to first-line therapy, survival after relapse is short (12 months). Standard therapy for recurrent MGMT-methylated glioblastoma is not standardized and may consist of re-resection, re-irradiation, and chemotherapy with temozolomide (TMZ), lomustine (CCNU), or a combination thereof. Preclinical results show that meclofenamate (MFA), originally developed as a nonsteroidal anti-inflammatory drug (NSAID) and registered in the USA, sensitizes glioblastoma cells to temozolomide-induced toxicity via inhibition of gap junction-mediated intercellular cytosolic traffic and demolishment of tumor microtube (TM)-based network morphology. METHODS: In this study, combined MFA/TMZ therapy will be administered (orally) in patients with first relapse of MGMT-methylated glioblastoma. A phase I component (6-12 patients, 2 dose levels of MFA + standard dose TMZ) evaluates safety and feasibility and determines the dose for the randomized phase II component (2 × 30 patients) with progression-free survival as the primary endpoint. DISCUSSION: This study is set up to assess toxicity and first indications of efficacy of MFA repurposed in the setting of a very difficult-to-treat recurrent tumor. The trial is a logical next step after the identification of the role of resistance-providing TMs in glioblastoma, and results will be crucial for further trials targeting TMs. In case of favorable results, MFA may constitute the first clinically feasible TM-targeted drug and therefore might bridge the idea of a TM-targeted therapeutic approach from basic insights into clinical reality. TRIAL REGISTRATION: EudraCT 2021-000708-39 . Registered on 08 February 2021.

NIX compensates lost role of parkin in cd-induced mitophagy in HeLa cells through phosphorylation.

Our previous study demonstrated that cadmium (Cd) is an effective inducer of mitophagy, which is mainly mediated by PINK1/Parkin pathway. However, the role of other mitophagy pathways in Cd-induced mitophagy remains elusive. The present study employed HeLa cells, lacking fully functional Parkin, as a cell model to study Parkin-independent mitophagy pathway induced by Cd. Our results showed that BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like (Bnip3L/NIX), an outer mitochondrial membrane mitophagy receptor, could provide an alternate pathway for Cd-induced mitophagy in HeLa cells. Specifically, 10 µM Cd for 12 h induced mitophagy in GM00637 and HeLa cells which was assessed by mitochondrial fusion to lysosomes and decreased expression of mitochondrial markers such as COX-IV and HSP60. Notably, in GM00637 cells, Cd-induced mitophagy was predominantly mediated by PINK1/Parkin pathway as evinced by translocation of Parkin to mitochondria. Interestingly, in HeLa cells, significant increase in NIX expression was occurred and mitophagy was induced under Cd exposure, suggesting NIX compensates lost role of Parkin in Cd-induced mitophagy in HeLa cells. These results were verified by knocking down NIX using siRNA in HeLa cells, which lead to abolished mitophagy process. Moreover, NIX phosphorylation at serine-81 significantly increased in cells treated with Cd implying that phosphorylation of NIX plays an important role in NIX-mediated mitophagy. These findings reveal a novel mechanism of Cd toxicity and suggest a compensatory role of NIX in Cd-induced mitophagy.

Ectopia associated MN1 fusions and aberrant activation in myeloid neoplasms with t(12;22)(p13;q12).

Chromosome translocation t(12;22)(p13;q12)/MN1-ETV6 and MN1 overexpression confer a subset of adverse prognostic AML but so far lack in-depth research. We focused on the clinical course and comprehensive genetic analysis of eight cases with t(12;22)(p13;q12) and one with t(12;17;22) (p13;q21;q13) to elucidate their molecular etiology and outcomes of allogeneic hemopoietic stem cell transplantation (allo-HSCT). The total incidence of t(12;22)(p13;q12) and related translocations was 0.32% in myeloid neoplasms. These patients were confirmed to have dismal prognosis when treated only with chemotherapy, and we firstly provided evidence that they can significantly benefit from timely allo-HSCT. Five cases were MN1-ETV6 positive, and a novel MN1-STAT3 fusion was identified in the patient with triadic translocation. Significant MN1 overexpression was observed in all three MN1-fusion-negative cases. Genetic analysis highlighted the evidence of an ectopic super-enhancer associated orchestrated mechanism of MN1 overexpression and ETV6 haploinsufficiency in t(12;22)(p13;q12) myeloid neoplasms, rather than the conventional thought of MN1-ETV6 fusion formation. We also disclosed the high concomitance of trisomy 8 and 531 Kbps focal 8q duplication in t(12;22)(p13;q12) cases. The new perspective about this entity of disease will enlighten further research to define the mechanism of tumorigenesis and discover effective treatments for MN1-driven malignancies.

Tissue inhibitor of matrix metalloproteinase-3 has both anti-metastatic and anti-tumourigenic properties.

TIMP-3 is one of four tissue inhibitors of matrix metalloproteinases, the endogenous inhibitors of the matrix metalloproteinase enzymes. These enzymes have an important role in metastasis, in the invasion of cancer cells through the basement membrane and extracellular matrix. TIMP-1, -2 and -4 both promote and inhibit tumour development, in a context-dependent manner, however TIMP-3 is consistently anti-tumourigenic. TIMP-3 is also the only insoluble member of the family, being either bound to the extracellular matrix or the low density lipoprotein-related protein-1, through which it can be endocytosed. Levels of TIMP-3 have also been shown to be regulated by micro RNAs and promoter hypermethylation, resulting in frequent silencing in many tumour types, to the extent that its expression has been suggested as a prognostic marker in some tumours, being associated with lower levels of metastasis, or better response to treatment. TIMP-3 has been shown to have anti-metastatic effects, both through inhibition of matrix metalloproteinases and ADAM family members and downregulation of angiogenesis. This occurs via interactions with receptors including VEGF, via modulation of signaling pathways and due to protease inhibition. TIMP-3 has also been shown to reduce tumour growth rate, most often by inducing apoptosis by stabilisation of death receptors. A number of successful mechanisms of delivery of TIMP-3 to tumour or inflammatory sites have been investigated in vitro or in animal studies. It may therefore be worthwhile further exploring the use of TIMP-3 as a potential anti-metastatic or anti-tumorigenic therapy for many tumour types.

OPCML: A Promising Biomarker and Therapeutic Avenue.

Opioid-binding protein/cell adhesion molecule (OPCML) is expressed in many tissues and localizes to the plasma membrane. It is a putative tumor suppressor that acts on receptor tyrosine kinases (RTKs), influencing cell cycle arrest, apoptosis, and migration. Its expression is silenced epigenetically in cancer, inferring potential diagnostic, prognostic, and therapeutic roles.

Non-viral nucleic acid delivery to the central nervous system and brain tumors.

Gene therapy is a rapidly emerging remedial route for many serious incurable diseases, such as central nervous system (CNS) diseases. Currently, nucleic acid medicines, including DNAs encoding therapeutic or destructive proteins, small interfering RNAs or microRNAs, have been successfully delivered to the CNS with gene delivery vectors using various routes of administration and have subsequently exhibited remarkable therapeutic efficiency. Among these vectors, non-viral vectors are favorable for delivering genes into the CNS as a result of their many special characteristics, such as low toxicity and pre-existing immunogenicity, high gene loading efficiency and easy surface modification. In this review, we highlight the main types of therapeutic genes that have been applied in the therapy of CNS diseases and then outline non-viral gene delivery vectors.

The discovery of shorter synthetic proteolytic peptides derived from Tob1 protein.

We screened nearly 1000 synthetic peptides and found that JAL-AK22 (KYEGHWYPEKPYKGSGFRCIHI), which is derived from the BoxA domain in the Tob1 protein, activates both unfolded and folded proMMP-7. Interestingly, the smaller derivative of JAL-AK22, termed JAL-TA9 (YKGSGFRMI) possessed auto-proteolytic activity and cleaved three synthetic peptides fragment (MMP18-33, MMP18-40, and Aß11-29) under physiological conditions. The kcat of JAL-TA9 was 4.58 × 10-4 min-1 against MMP18-33 and 6.5 × 10-4 min-1 against MMP18-40. These kinetic parameters are lower than those of general proteinases like trypsin, for which the kcat is 247.2 × 105 min-1 against benzoyl-l-arginine ethyl ester. In addition, a 5-mer peptide derived from JAL-TA9, GSGFR also cleaved Aß11-29. These proteolytic activities were inhibited by AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride), a serine protease inhibitor. Our results demonstrate that some small synthetic peptides have protease activity. Thus, we propose calling small peptides possessing with protease activity Catalytides (catalytic peptides). We expect that our findings will stimulate the development of novel Catalytides and related applications such as the development of strategic peptide drugs.

[The anti-cancer effect of ZR30 protein via targeting extracellular signal proteins of different cell subpopulations of glioma].

Objective: To investigate the roles and anti-cancer mechanism of artificially synthesized EGF-containing fibulin-like extracellular matrix protein (EFEMP1) derived tumor suppressor ZR30 protein in glioma (GBM). Methods: ZR30 protein were in vitro expressed using a wheat germ cell-free system. GBM cell lines (U251, U251NS, and U87) were cultured for 2-3 days in the presence or absence of ZR30 treatment. MMP-2 level was detected by gelatin zymography assay, moreover, the expression of EGFR, Notch-1 and p-Akt/Akt levels were determined by western blot. Additionally, MTT assay was used to measure ZR30's effect on the cell proliferation of U251 and U251NS cells. Furthermore, pre-mixed U251-GFP and U251NS-RFP cells (1∶9) were injected into the brain of nude mice, and then ZR30 or PBS was injected into the intra-tumor after 10 and 21 days, respectively. Then DNA was extracted from the right brain of nude mice in each group. Comparative quantitative polymerase chain reaction (CQ-PCR) was used to examine the copy numbers of human gene hSPAG16, mouse gene mSpag16, GFP and RFP. The survival status of each group of nude mice was also observed. Results: The levels of activated MMP-2 in U87 and U251 cells were lower after 10, 50 and 100 ng/ml ZR30 treatment for 2-3 days. Western blot analysis showed that ZR30 treatment reduced the expression of EGFR, Notch-1 and p-Akt/Akt in U251 cells, and inhibited Notch-1 and p-Akt/Akt expression in U251NS cells, and then decreased the response of U251 cells to EGF stimulation. Moreover, ZR30 inhibited the cell proliferation of U251 and U251NS two days after exposure. The in vivo orthotopic GBM xenografts were successfully constructed. CQ-PCR results indicated that the hSPAG16/mSpag16 ratios of mice in PBS group and ZR30 treatment groups at 180, 700, and 1 800 ng dosages were 3.67±2.82, 1.18±0.97, 1.75±1.55 and 1.38±1.17, respectively, and ZR30 treatment groups showed significantly lower ratios than the PBS group (P<0.05 for all). Correspondingly, the ratios of GFP/RFP in each group were 1.97±0.80, 1.97±0.85, 1.48±0.71 and 1.73±0.77, respectively, showing no statistical significance (P>0.05 for all). When treatment was performed 10 d after cell implantation, and the median survival time of mice in PBS group and ZR30 group was 40.5 days and 59.0 days, respectively. When treatment was performed 21 d after cell implantation, the median survival time of mice in PBS group and ZR30 group was extended to 57.0 days and 74.5 days, respectively. The median survival time of ZR30 treatment groups significantly prolonged (P<0.05 for all). Conclusions: ZR30 inhibits in vitro cell growth, invasion, angiogenesis and stemness maintenance in glioma via suppressing activated MMP-2, EGFR, p-Akt/Akt and Notch-1 proteins. In vivo, ZR30 markedly increased survival of mice harboring glioma xenografts, even for only one intra-tumoral injection at the time of early tumor formation. Overall, the in vivo and in vitro experiments supported the therapeutic potential of ZR30 for GBM.

Peripheral Gene Therapeutic Rescue of an Olfactory Ciliopathy Restores Sensory Input, Axonal Pathfinding, and Odor-Guided Behavior.

Cilia of olfactory sensory neurons (OSNs) are the primary site of odor binding; hence, their loss results in anosmia, a clinical manifestation of pleiotropic ciliopathies for which there are no curative therapies. We used OSN-specific Ift88 knock-out mice (Ift88osnKO) of both sexes to examine the mechanisms of ciliopathy-induced olfactory dysfunction and the potential for gene replacement to rescue odorant detection, restore olfactory circuitry, and restore odor-guided behaviors. Loss of OSN cilia in Ift88osnKO mice resulted in substantially reduced odor detection and odor-driven synaptic activity in the olfactory bulb (OB). Defects in OSN axon targeting to the OB were also observed in parallel with aberrant odor-guided behavior. Intranasal gene delivery of wild-type IFT88 to Ift88osnKO mice rescued OSN ciliation and peripheral olfactory function. Importantly, this recovery of sensory input in a limited number of mature OSNs was sufficient to restore axonal targeting in the OB of juvenile mice, and with delayed onset in adult mice. In addition, restoration of sensory input re-established course odor-guided behaviors. These findings highlight the spare capacity of the olfactory epithelium and the plasticity of primary synaptic input into the central olfactory system. The restoration of peripheral and central neuronal function supports the potential for treatment of ciliopathy-related anosmia using gene therapy.SIGNIFICANCE STATEMENT Ciliopathies, for which there are no curative therapies, are genetic disorders that alter cilia morphology and/or function in numerous tissue types, including the olfactory system, leading to sensory dysfunction. We show that in vivo intranasal gene delivery restores peripheral olfactory function in a ciliopathy mouse model, including axonal targeting in the juvenile and adult olfactory bulb. Gene therapy also demonstrated restoration of olfactory perception by rescuing odor-guided behaviors. Understanding the therapeutic window and viability for gene therapy to restore odor detection and perception may facilitate translation of therapies to ciliopathy patients with olfactory dysfunctions.

Clinical Trial of MGMT(P140K) Gene Therapy in the Treatment of Pediatric Patients with Brain Tumors.

Gene transfer targeting hematopoietic stem cells (HSC) in children has shown sustained therapeutic benefit in the treatment of genetic diseases affecting the immune system, most notably in severe combined immunodeficiencies affecting T-cell function. The HSC compartment has also been successfully targeted using gene transfer in children with genetic diseases affecting the central nervous system, such as metachromatic leukodystrophy and adrenoleukodystrophy. HSCs are also a target for genetic modification in strategies aiming to confer drug resistance to chemotherapy agents so as to reduce off-target toxicity, and to allow for chemotherapy dose escalation with the possibility of enhanced therapeutic benefit. In a trial of this strategy in adult glioma patients, significant engraftment of gene-modified HSCs expressing a mutant of the DNA repair protein O6-methyl-guanine-methyl-transferase (MGMT(P140K)) showed potential in conferring drug resistance against the combined effect of O6-benzylguanine (O6BG)/temozolomide (TMZ) chemotherapy. The aim was to test the safety and feasibility of this approach in children with poor prognosis brain tumors. In this Phase I trial, seven patients received gene-modified HSC following myelo-suppressive conditioning, but with only transient low-level engraftment of MGMT(P140K) gene-modified cells detectable in four patients. All patients received O6BG/TMZ chemotherapy following infusion of gene-modified cells, with five patients eligible for chemotherapy dose escalation, though in the absence of demonstrable transgene-mediated chemoprotection. Since all gene-modified cell products met the criteria for release and assays for engraftment potential met expected outcome measures, inadequate cell dose, conditioning chemotherapy, and/or underlying bone-marrow function may have contributed to the lack of sustained engraftment of gene-modified cells. We were able to demonstrate safe conduct of a technically complex Phase I study encompassing manufacture of the gene therapy vector, genetically modified cells, and a drug product specifically for the trial in compliance with both local and national regulatory requirements.

Netrin-1 in cancer: Potential biomarker and therapeutic target?

Netrin-1, a laminin-related protein, is known to be involved in the nervous system development. Recently, Netrin-1's involvement in other processes such as cell adhesion, motility, proliferation, and differentiation that are important for the development of epithelial tissues has been described. In addition, Netrin-1 and its receptors, deleted in colorectal cancer and uncoordinated-5 homolog, have been linked to apoptosis and angiogenesis. Since these properties are essential for tumor development, Netrin-1 and its receptors have been reported to promote tumorigenesis in many types of cancers. Here, we review the Netrin-1 mediated regulation of cancer, its potential use as a biomarker, and the targeting of the Netrin-1 pathway to treat cancers.