The BlueScreen HC assay to predict the genotoxic potential of fragrance materials.

BlueScreen HC is a mammalian cell-based assay for measuring the genotoxicity and cytotoxicity of chemical compounds and mixtures. The BlueScreen HC assay has been utilized at the Research Institute for Fragrance Materials in a safety assessment program as a screening tool to prioritize fragrance materials for higher-tier testing, as supporting evidence when using a read-across approach, and as evidence to adjust the threshold of toxicological concern. Predictive values for the BlueScreen HC assay were evaluated based on the ability of the assay to predict the outcome of in vitro and in vivo mutagenicity and chromosomal damage genotoxicity assays. A set of 371 fragrance materials was assessed in the BlueScreen HC assay along with existing or newly generated in vitro and in vivo genotoxicity data. Based on a weight-of-evidence approach, the majority of materials in the data set were deemed negative and concluded not to have the potential to be genotoxic, while only a small proportion of materials were determined to show genotoxic effects in these assays. Analysis of the data set showed a combination of high positive agreement but low negative agreement between BlueScreen HC results, in vitro regulatory genotoxicity assays, and higher-tier test results. The BlueScreen HC assay did not generate any false negatives, thereby providing robustness when utilizing it as a high-throughput screening tool to evaluate the large inventory of fragrance materials. From the perspective of protecting public health, it is desirable to have no or minimal false negatives, as a false-negative result may incorrectly indicate the lack of a genotoxicity hazard. However, the assay did have a high percentage of false-positive results, resulting in poor positive predictivity of the in vitro genotoxicity test battery outcome. Overall, the assay generated 100% negative predictivity and 3.9% positive predictivity. In addition to the data set of 371 fragrance materials, 30 natural complex substances were evaluated for BlueScreen HC, Ames, and in vitro micronucleus assay, and a good correlation in all three assays was observed. Overall, while a positive result may have to be further investigated, these findings suggest that the BlueScreen HC assay can be a valuable screening tool to detect the genotoxic potential of fragrance materials and mixtures.

A Cautionary tale for using read-across for cancer hazard classification: Case study of isoeugenol and methyl eugenol.

Chemical grouping and read-across are frequently used non-animal alternatives for filling toxicological data gaps. When grouping chemicals, it is critical to define the applicability domain because minor differences in chemical structure can lead to significant differences in toxicity. Here, we present a case study on isoeugenol and methyl eugenol, which are scheduled for review by IARC in June 2023, to illustrate that structural similarity alone may not be sufficient to group chemicals for hazard classification. Isoeugenol and methyl eugenol are plant-derived phenylpropenes that share similar physicochemical properties. The major metabolic pathway for isoeugenol includes conjugation of the phenolic hydroxyl group with sulfate and glucuronic acid as an efficient detoxification process, whereas the major metabolic pathway for methyl eugenol involves benzylic hydroxylation and formation of the 1'-sulfoxymethyleugenol which leads to carbocation formation. The carbocation can form DNA adducts and induce genotoxicity and carcinogenicity. Consistently, genotoxicity and carcinogenicity alerts are identified from in silico prediction tools for methyl eugenol but not isoeugenol. Moreover, the available toxicogenomic, genotoxicity, and carcinogenicity studies confirm that these chemicals have significantly different bioactivities. Data on other structurally similar chemicals further supports our conclusion that it is not appropriate to group these two chemicals for cancer hazard classification.

Clinical Performance and Parental Satisfaction with Composite Strip Crown and Prefabricated Zirconia Crown for Primary Anterior Teeth: A Randomized Clinical Trial.

AIM: To compare clinical performance and parental satisfaction with composite strip crown and prefabricated zirconia crown for primary anterior teeth. MATERIALS AND METHODS: The study compares clinical evaluation and parental satisfaction of two different crowns for primary anterior teeth. A total of 102 teeth in each group selected between ages 3 and 6 years, who met the inclusion criteria, were randomly allocated into two groups for further evaluation. Group A for strip crowns (55 teeth) and group B for zirconia crowns (47 teeth). The crowns were evaluated clinically with various criteria like-color match, crown retention, gingival health, crown contour, opposing tooth wear, marginal integrity, and recurrent caries. The samples were also evaluated for parental satisfaction based on 5-point Likert scale and child liking was also recorded with Smiley face Likert scale at baseline, 3 and 9 months. Statistical analysis was done using Chi-square test (p <0.05). RESULTS: Zirconia crowns showed better color match, crown retention, crown contour, and gingival health. Strip crowns showed more discoloration and chipping of material over a period of time. None of the samples showed opposing tooth wear, open margins, and recurrent caries in strip and zirconia crown group. Parents and children both were highly satisfied with zirconia crowns. CONCLUSION: Clinically zirconia crowns showed higher success rate as compared to strip crowns and parental overall satisfaction was higher for zirconia crowns. CLINICAL SIGNIFICANCE: Zirconia crowns exhibited a higher clinical performance and parental satisfaction; hence, if affordability is out weighted, zirconia crown stands better with esthetics of the child.

Capillary microsampling in mice: effective way to move from sparse sampling to serial sampling in pharmacokinetics profiling.

Pharmacokinetic studies are an integral part of drug discovery and development. Mice are the commonly used species for pharmacokinetics studies during early discovery studies. Conventionally, composite PK profiles are obtained from mice studies due to the physiological limitations of the total blood volume that can be drawn over a certain period.With advancements in bioanalytical instrumentation and in blood sampling techniques, analysis with small volume (<50 µL) became feasible enabling serial blood sampling from the mouse for PK studies. The objective of the current study was to develop and establish a serial blood sampling technique in mouse and compare it with the conventional sparse sampling method (composite PK) following oral administration of widely used NSAIDs, diclofenac, celecoxib and tenoxicam, into Swiss Albino mice.The pharmacokinetic parameters of all three probe drugs by serial blood sampling were comparable with that of sparse sampling method. There was no significant difference between the whole blood concentration time profiles of all three drugs between serial sampling and sparse sampling suggesting serial blood sampling method can be easily implemented for mice PK studies.Serial blood sampling technique requires use of fewer number of animals, less quantity of test compound and reduces the possible dosing errors as fewer number of animals need to be dosed resulting in quality PK data and enabling comparison of inter-animal differences in PK profile.

Bolstering the existing database supporting the non-cancer Threshold of Toxicological Concern values with toxicity data on fragrance-related materials.

The use of threshold of toxicological concern (TTC) supports the safety assessment of exposure to low levels of chemicals when toxicity data are limited. The Research Institute for Fragrance Materials (RIFM) delivers safety assessments for fragrance materials that result in safe products for consumer use. A major goal for the RIFM safety assessment program is to invest in alternative methods to animal testing for use in assessment of fragrance materials. This includes use of TTC, which provides a pragmatic approach for safety evaluation of fragrance materials in the absence of chemical-specific toxicity data and reduces the need to generate new animal data. To bolster the TTC approach for support of fragrance materials and specifically to strengthen the Cramer class II threshold, the RIFM database was reviewed with a goal of identifying fragrance materials with data that can be added to the existing TTC databases. The RIFM database identified a total of 476 chemicals that were added to the existing TTC databases. The chemicals were then individually assigned a Cramer class and 238, 76 and 162 chemicals in Cramer class I, II and III respectively were identified. The RIFM-TTC dataset was then combined with the COSMOS-Federated TTC dataset for a total of 421, 111 and 795 chemicals in Cramer class I, II and III respectively. The combined dataset further expands the chemical space thereby providing more robust 5th percentile thresholds. Moreover, the combined dataset bolsters the threshold for Cramer class II to include a total of 111 chemicals which is an improvement over the original (Munro) TTC dataset which only included 28 chemicals in Cramer Class II and the COSMOS Federated dataset which had 40 chemicals. This allows for a more reliable and robust 5th percentile NOAEL value for Cramer class II chemicals of 1.27 mg/kg bw/day. The 5th percentile NOAELs for Cramer class I, II and III from the combined dataset are 4.91, 1.27 and 0.29 mg/kg bw/day, which supports the threshold values derived from the original Munro dataset. This work confirms the adequacy of the existing TTC values and provides further support for the use of TTC as a tool to conduct safety assessments for fragrance materials. It further opens the future possibility of updating the existing values with more robust TTC values for fragrance and cosmetic materials.

Discovery and evaluation of novel FAAH inhibitors in neuropathic pain model.

Conceptual design and modification of urea moiety in chemotype PF-3845/04457845, the bench marking irreversible inhibitor of fatty acid amide hydrolase (FAAH), led to discovery of a novel nicotinamide-based lead 12a having reversible mechanism of action. Focused SAR around the pyridine heterocycle (Ar) in 12a (Tables 1 and 2) resulted into four shortlisted compounds, (-)-12a, (-)-12i, (-)-12l-m. The required (-)-enantiomers were obtained via diastereomeric resolution of a novel chiral dissymmetric intermediate 15. Based on comparative profile of FAAH potency, metabolic stability in liver microsome, liability of inhibiting major hCYP450 isoforms, rat PK, and brain penetration ability, two SAR optimized compounds, (-)-12l and (-)-12m, were selected for efficacy study in rat model of chemotherapy-induced peripheral neuropathy (CIPN). Both the compounds exhibited dose related antihyperalgesic effects, when treated with 3-30 mg/kg po for 7 days. The effects at 30 mg/kg are comparable to that of PF-04457845 (10 mg/kg) and Tramadol (40 mg/kg).

Evaluation of separate role of intestine and liver in first pass metabolism of budesonide in rat.

1. Budesonide, a potent topical corticosteroid, reported to have low oral bioavailability in mice, rat, dog and human due to rapid first pass metabolism. However, there is insufficient information available in literature regarding the role of intestine and or liver responsible for the first pass metabolism of budesonide. 2. Current study in rats investigates the role of intestine and liver in first pass metabolism of budesonide using two in vivo models. Additionally, budesonide was also evaluated in in vitro assays such as thermodynamic solubility, permeability in Caco-2 cells and stability in simulated gastric (SGF), intestinal fluids (SIF) to understand the underlaying cause for low oral bioavailability. 3. Budesonide showed low oral, intra-duodenal and high intra-portal bioavailability in rat. In a dual vein cannulated rat model, intestinal and hepatic extraction ratios calculated based upon intestinal availability (Fa·Fg) and hepatic availability (Fh), suggests hepatic extraction of budesonide is minimal compared to intestinal. 4. In vitro results suggest, solubility and permeability may not be a barrier for the observed low oral bioavailability in rats. 5. Correlating the in vitro and in vivo data together, it can be concluded that, intestine might be playing major role in first pass metabolism of budesonide.

Cardiotoxic (Arrhythmogenic) Effects of 1,1-Difluoroethane Due to Electrolyte Imbalance and Cardiomyocyte Damage.

Inhalant abuse is the intentional inhalation of chemical vapors to attain euphoric effects. Many common household products are abused by inhalation and one is 1,1-difluoroethane (DFE), which is a halogenated hydrocarbon used in refrigeration, dust-off spray, and airbrush painting. Although many human DFE abuse cases have been studied, the etiology and mechanism of sudden death is still unknown. In this study, an animal model was used to simulate the human conditions of DFE inhalation abuse that results in sudden death.Current research targets mechanistic studies involving electrolyte changes and cardiomyocyte damage after DFE administration in vivo. To investigate these changes, Sprague Dawley rats (N = 6) were exposed to 30 seconds of 20 L/min of DFE in multiple doses. Isoflurane acted as a control. Two additional groups, epinephrine and epinephrine + DFE, were included to simulate the clinical condition of DFE abuse. Plasma sodium, potassium, calcium, and magnesium levels were measured, followed by lactate dehydrogenase, creatine kinase, and cardiac troponin I levels. In addition, oxidative stress markers were also evaluated in all animal groups. Electrolyte levels showed a significant rise in plasma potassium and magnesium levels for the treated groups. In addition, lactate dehydrogenase, creatine kinase, and cardiac troponin I levels in DFE and epinephrine + DFE administered rats were significantly elevated as compared with control. Some oxidative stress makers were also elevated significantly in treatment groups. Furthermore, histopathological analysis showed hyperemia/congestion in treated rats.These results support cardiotoxic effects indicating that DFE results in fatal arrhythmias, and the study can be important during clinical cases involving inhalant abuse.

Mesenchymal glioma stem cells are maintained by activated glycolytic metabolism involving aldehyde dehydrogenase 1A3.

Tumor heterogeneity of high-grade glioma (HGG) is recognized by four clinically relevant subtypes based on core gene signatures. However, molecular signaling in glioma stem cells (GSCs) in individual HGG subtypes is poorly characterized. Here we identified and characterized two mutually exclusive GSC subtypes with distinct dysregulated signaling pathways. Analysis of mRNA profiles distinguished proneural (PN) from mesenchymal (Mes) GSCs and revealed a pronounced correlation with the corresponding PN or Mes HGGs. Mes GSCs displayed more aggressive phenotypes in vitro and as intracranial xenografts in mice. Further, Mes GSCs were markedly resistant to radiation compared with PN GSCs. The glycolytic pathway, comprising aldehyde dehydrogenase (ALDH) family genes and in particular ALDH1A3, were enriched in Mes GSCs. Glycolytic activity and ALDH activity were significantly elevated in Mes GSCs but not in PN GSCs. Expression of ALDH1A3 was also increased in clinical HGG compared with low-grade glioma or normal brain tissue. Moreover, inhibition of ALDH1A3 attenuated the growth of Mes but not PN GSCs. Last, radiation treatment of PN GSCs up-regulated Mes-associated markers and down-regulated PN-associated markers, whereas inhibition of ALDH1A3 attenuated an irradiation-induced gain of Mes identity in PN GSCs. Taken together, our data suggest that two subtypes of GSCs, harboring distinct metabolic signaling pathways, represent intertumoral glioma heterogeneity and highlight previously unidentified roles of ALDH1A3-associated signaling that promotes aberrant proliferation of Mes HGGs and GSCs. Inhibition of ALDH1A3-mediated pathways therefore might provide a promising therapeutic approach for a subset of HGGs with the Mes signature.

Tumor-specific activation of the C-JUN/MELK pathway regulates glioma stem cell growth in a p53-dependent manner.

Accumulated evidence suggests that glioma stem cells (GSCs) may contribute to therapy resistance in high-grade glioma (HGG). Although recent studies have shown that the serine/threonine kinase maternal embryonic leucine-zipper kinase (MELK) is abundantly expressed in various cancers, the function and mechanism of MELK remain elusive. Here, we demonstrate that MELK depletion by shRNA diminishes the growth of GSC-derived mouse intracranial tumors in vivo, induces glial fibrillary acidic protein (+) glial differentiation of GSCs leading to decreased malignancy of the resulting tumors, and prolongs survival periods of tumor-bearing mice. Tissue microarray analysis with 91 HGG tumors demonstrates that the proportion of MELK (+) cells is a statistically significant indicator of postsurgical survival periods. Mechanistically, MELK is regulated by the c-Jun NH(2)-terminal kinase (JNK) signaling and forms a complex with the oncoprotein c-JUN in GSCs but not in normal progenitors. MELK silencing induces p53 expression, whereas p53 inhibition induces MELK expression, indicating that MELK and p53 expression are mutually exclusive. Additionally, MELK silencing-mediated GSC apoptosis is partially rescued by both pharmacological p53 inhibition and p53 gene silencing, indicating that MELK action in GSCs is p53 dependent. Furthermore, irradiation of GSCs markedly elevates MELK mRNA and protein expression both in vitro and in vivo. Clinically, recurrent HGG tumors following the failure of radiation and chemotherapy exhibit a statistically significant elevation of MELK protein compared with untreated newly diagnosed HGG tumors. Together, our data indicate that GSCs, but not normal cells, depend on JNK-driven MELK/c-JUN signaling to regulate their survival, maintain GSCs in an immature state, and facilitate tumor radioresistance in a p53-dependent manner.

MELK-dependent FOXM1 phosphorylation is essential for proliferation of glioma stem cells.

Glioblastoma multiforme (GBM) is a life-threatening brain tumor. Accumulating evidence suggests that eradication of glioma stem-like cells (GSCs) in GBM is essential to achieve cure. The transcription factor FOXM1 has recently gained attention as a master regulator of mitotic progression of cancer cells in various organs. Here, we demonstrate that FOXM1 forms a protein complex with the mitotic kinase MELK in GSCs, leading to phosphorylation and activation of FOXM1 in a MELK kinase-dependent manner. This MELK-dependent activation of FOXM1 results in a subsequent increase in mitotic regulatory genes in GSCs. MELK-driven FOXM1 activation is regulated by the binding and subsequent trans-phosphorylation of FOXM1 by another kinase PLK1. Using mouse neural progenitor cells (NPCs), we found that transgenic expression of FOXM1 enhances, while siRNA-mediated gene silencing diminishes neurosphere formation, suggesting that FOXM1 is required for NPC growth. During tumorigenesis, FOXM1 expression sequentially increases as cells progress from NPCs, to pretumorigenic progenitors and GSCs. The antibiotic Siomycin A disrupts MELK-mediated FOXM1 signaling with a greater sensitivity in GSC compared to neural stem cell. Treatment with the first-line chemotherapy agent for GBM, Temozolomide, paradoxically enriches for both FOXM1 (+) and MELK (+) cells in GBM cells, and addition of Siomycin A to Temozolomide treatment in mice harboring GSC-derived intracranial tumors enhances the effects of the latter. Collectively, our data indicate that FOXM1 signaling through its direct interaction with MELK regulates key mitotic genes in GSCs in a PLK1-dependent manner and thus, this protein complex is a potential therapeutic target for GBM.

Clinical Assessment of Preemptive Analgesia on Success of Pulpal Anesthesia and Postendodontic Pain in Children with Irreversible Pulpitis: A Randomized Comparative Study.

Introduction: Optimal pain management of symptomatic pulpitis in formative years goes a long way in developing a positive dental attitude. Efforts should be made to increase the success of anesthesia, thus diminishing negative dental experiences. The aim of the study was to assess the efficacy of preemptive analgesia on the success of pulpal anesthesia following inferior alveolar nerve block (IANB) in children with symptomatic irreversible pulpitis and on reducing postendodontic pain. Materials and methods: The research design was an in vivo, three-group, parallel, quadruple-blind study. A total of 75 patients were randomly allocated to one of the three groups-group I: ibuprofen, group II: combination of ibuprofen and paracetamol, and group III: multivitamin (placebo). Premedication was given 45 minutes before treatment, and patients received IANB in a standardized manner. Pain during pulpectomy was recorded using the face, legs, activity, cry, consolability (FLACC) scale and postoperatively using Wong-Baker's pain rating scale (WBPRS) at 4, 12, and 24 hours. Success was measured if the pain felt was of no or mild intensity. Results: Success of IANB was 64% for ibuprofen, 72% for the combination group, and 40% for the placebo group, with no statistically significant difference between all groups (p = 0.06) on the FLACC scale. At 4 hours postoperatively, a significant difference (p = 0.02) was found among groups with more children experiencing no or mild pain in groups I and II and the highest number of rescue medications taken by the placebo group. Conclusion: Ibuprofen and a combination of ibuprofen and acetaminophen as preemptive analgesics had no significant effect on the success rate of IANB, although it was effective in reducing pain at 4 hours postoperatively. How to cite this article: Gori NA, Patel MC, Bhatt RK, et al. Clinical Assessment of Preemptive Analgesia on Success of Pulpal Anesthesia and Postendodontic Pain in Children with Irreversible Pulpitis: A Randomized Comparative Study. Int J Clin Pediatr Dent 2024;17(1):72-78.

A comparative evaluation of light cure calcium silicate and resin-modified glass ionomer as indirect pulp capping agent in primary molars: A randomized clinical trial.

Background: Minimally invasive techniques should be employed to maintain pulp vitality without affecting physiological tooth resorption for pediatric patients. This study aims to evaluate clinical and radiographic success of light cure calcium silicate and resin-modified glass ionomer (RMGI) as indirect pulp-capping agent in primary molars in children between 5 and 9 years. Materials and Methods: In this randomized clinical trial study, 40 primary molars with the International Caries Detection and Assessment System criteria 4-6 score were divided randomly into two groups after computerized randomization method. After caries excavation by minimally invasive dentistry principle, affected dentin was lined by either light cure calcium silicate (Group I) or RMGI (Group II) liner followed by composite restoration. Clinical success was determined with the absence of symptoms and bitewing radiographs were taken at baseline, 3, and 6 months' interval to measure increase in dentin increment using ImageJ software. Statistical analysis for intergroup comparison was done using Paired t-test, and Independent t-test was used for intragroup comparison. The level of statistical significance was set at P < 0.05. Results: Change in dentin increment seen from baseline to 6 months in Group I was 0.19 mm and in Group II was 0.20 mm (P = 0.924). Intragroup increment was statistically significant during all follow-up intervals for both the groups (Group I - P ≤ 0.001, Group II - P = 0.009). For baseline remaining dentin thickness >1.5 mm, statistically significant increase was observed in the dentin increment at 3 months' interval between both the groups. Conclusion: Both TheraCal LC and Vitrebond show acceptable clinical and radiographic results when used in primary molars as indirect pulp treatment agents. Both the liners are equivalent to each other in terms of feasibility and cost-effectiveness but TheraCal LC can be preferred due to better handling and avoiding the manipulation step thereby reducing treatment time.

Clinical and Radiographic Evaluation of Different Glass Ionomer Restorative Materials in Primary Molars: A Comparative Randomized Clinical Trial.

Aim: The study aimed to evaluate and compare the clinical and radiographic success rate of Ketac Universal (3M), GC 9 EXTRA (GC), and Cention N (Ivoclar) restorative materials in primary molars. Materials and methods: The study was conducted as randomized clinical trial in children aged 3-8 years old, out of which a total of 75 primary molars were selected in 43 patients who met the inclusion criteria. The sample size was further divided into three groups of restorative materials, which included group I-Ketac Universal (n = 25), group II-GC 9 EXTRA (n = 25), and group III-Cention N (n = 25). Class I restorations were placed randomly according to the computerized randomization in primary molars and evaluated at baseline (1 week), 6 months, and 12 months according to modified United States Public Health Service (USPHS) criteria and bitewing radiographs. Results: On intercomparison of three groups at 12 months, there was no significant difference found, while on pairwise comparison of Ketac Universal and Cention N, there was a significant difference in relation to surface texture (p = 0.04*) and anatomic contour (p = 0.04*) at 12 months. Conclusion: Newly introduced restorative material Cention N exhibited improved physical and mechanical properties and can be recommended as a cost-effective restorative and easy-to-use material in posterior load-bearing primary molars. Clinical significance: Restoration of primary teeth continues to be an important aspect of restorative dentistry. The longevity of restorations in primary teeth is significantly different for all materials compared to permanent dentition. This makes the assessment of these restorations as a separate group meaningful. How to cite this article: Kataria VG, Patel MC, Bhatt R, et al. Clinical and Radiographic Evaluation of Different Glass Ionomer Restorative Materials in Primary Molars: A Comparative Randomized Clinical Trial. Int J Clin Pediatr Dent 2023;16(6):829-836.

Topical SCD-153, a 4-methyl itaconate prodrug, for the treatment of alopecia areata.

Alopecia areata is a chronic hair loss disorder that involves autoimmune disruption of hair follicles by CD8+  T cells. Most patients present with patchy hair loss on the scalp that improves spontaneously or with topical and intralesional steroids, topical minoxidil, or topical immunotherapy. However, recurrence of hair loss is common, and patients with extensive disease may require treatment with oral corticosteroids or oral Janus kinase (JAK) inhibitors, both of which may cause systemic toxicities with long-term use. Itaconate is an endogenous molecule synthesized in macrophages that exerts anti-inflammatory effects. To investigate the use of itaconate derivatives for treating alopecia areata, we designed a prodrug of 4-methyl itaconate (4-MI), termed SCD-153, with increased lipophilicity compared to 4-MI (CLogP 1.159 vs. 0.1442) to enhance skin and cell penetration. Topical SCD-153 formed 4-MI upon penetrating the stratum corneum in C57BL/6 mice and showed low systemic absorption. When added to human epidermal keratinocytes stimulated with polyinosinic-polycytidylic acid (poly I:C) or interferon (IFN)γ, SCD-153 significantly attenuated poly I:C-induced interleukin (IL)-6, Toll-like receptor 3, IL-1ß, and IFNß expression, as well as IFNγ-induced IL-6 expression. Topical application of SCD-153 to C57BL/6 mice in the resting (telogen) phase of the hair cycle induced significant hair growth that was statistically superior to vehicle (dimethyl sulfoxide), the less cell-permeable itaconate analogues 4-MI and dimethyl itaconate, and the JAK inhibitor tofacitinib. Our results suggest that SCD-153 is a promising topical candidate for treating alopecia areata.

EZH2 protects glioma stem cells from radiation-induced cell death in a MELK/FOXM1-dependent manner.

Glioblastoma (GBM)-derived tumorigenic stem-like cells (GSCs) may play a key role in therapy resistance. Previously, we reported that the mitotic kinase MELK binds and phosphorylates the oncogenic transcription factor FOXM1 in GSCs. Here, we demonstrate that the catalytic subunit of Polycomb repressive complex 2, EZH2, is targeted by the MELK-FOXM1 complex, which in turn promotes resistance to radiation in GSCs. Clinically, EZH2 and MELK are coexpressed in GBM and significantly induced in postirradiation recurrent tumors whose expression is inversely correlated with patient prognosis. Through a gain-and loss-of-function study, we show that MELK or FOXM1 contributes to GSC radioresistance by regulation of EZH2. We further demonstrate that the MELK-EZH2 axis is evolutionarily conserved in Caenorhabditis elegans. Collectively, these data suggest that the MELK-FOXM1-EZH2 signaling axis is essential for GSC radioresistance and therefore raise the possibility that MELK-FOXM1-driven EZH2 signaling can serve as a therapeutic target in irradiation-resistant GBM tumors.

Crosstalk between glioma-initiating cells and endothelial cells drives tumor progression.

Glioma-initiating cells (GIC), which reside within the perivascular microenvironment to maintain self-renewal capacity, are responsible for glioblastoma initiation, progression, and recurrence. However, the molecular mechanisms controlling crosstalk between GICs and endothelial cells are poorly understood. Here, we report that, in both GICs and endothelial cells, platelet-derived growth factor (PDGF)-driven activation of nitric oxide (NO) synthase increases NO-dependent inhibitor of differentiation 4 (ID4) expression, which in turn promotes JAGGED1-NOTCH activity through suppression of miR129 that specifically represses JAGGED1 suppression. This signaling axis promotes tumor progression along with increased GIC self-renewal and growth of tumor vasculature in the xenograft tumors, which is dramatically suppressed by NOTCH inhibitor. ID4 levels correlate positively with NOS2 (NO synthase-2), HES1, and HEY1 and negatively with miR129 in primary GICs. Thus, targeting the PDGF-NOS-ID4-miR129 axis and NOTCH activity in the perivascular microenvironment might serve as an efficacious therapeutic modality for glioblastoma.

Multi-kinase inhibitor C1 triggers mitotic catastrophe of glioma stem cells mainly through MELK kinase inhibition.

Glioblastoma multiforme (GBM) is a highly lethal brain tumor. Due to resistance to current therapies, patient prognosis remains poor and development of novel and effective GBM therapy is crucial. Glioma stem cells (GSCs) have gained attention as a therapeutic target in GBM due to their relative resistance to current therapies and potent tumor-initiating ability. Previously, we identified that the mitotic kinase maternal embryonic leucine-zipper kinase (MELK) is highly expressed in GBM tissues, specifically in GSCs, and its expression is inversely correlated with the post-surgical survival period of GBM patients. In addition, patient-derived GSCs depend on MELK for their survival and growth both in vitro and in vivo. Here, we demonstrate evidence that the role of MELK in the GSC survival is specifically dependent on its kinase activity. With in silico structure-based analysis for protein-compound interaction, we identified the small molecule Compound 1 (C1) is predicted to bind to the kinase-active site of MELK protein. Elimination of MELK kinase activity was confirmed by in vitro kinase assay in nano-molar concentrations. When patient-derived GSCs were treated with C1, they underwent mitotic arrest and subsequent cellular apoptosis in vitro, a phenotype identical to that observed with shRNA-mediated MELK knockdown. In addition, C1 treatment strongly induced tumor cell apoptosis in slice cultures of GBM surgical specimens and attenuated growth of mouse intracranial tumors derived from GSCs in a dose-dependent manner. Lastly, C1 treatment sensitizes GSCs to radiation treatment. Collectively, these data indicate that targeting MELK kinase activity is a promising approach to attenuate GBM growth by eliminating GSCs in tumors.

Blockade of EGFR signaling promotes glioma stem-like cell invasiveness by abolishing ID3-mediated inhibition of p27(KIP1) and MMP3 expression.

Aberrant epidermal growth factor receptor (EGFR) signaling is a typical oncogenic signature in glioblastoma. Here, we show that EGFR inhibition in primary glioma stem cells (GSCs) with oncogenic EGFRvIII and EGFRvIII-transduced glioma stem-like cells promotes invasion by decreasing ID3 levels. ID3 suppresses GSC invasiveness by inhibiting p27(KIP1)-RhoA-dependent migration and MMP3 expression. Xenograft and human glioblastoma specimens show that ID3 localizes within glioblastoma cores, whereas p27(KIP1) and MMP3 are predominantly expressed in glioma cells in invasive fronts. Together, our findings show that EGFR inhibition induces GSC invasiveness by abolishing ID3-mediated inhibition of p27(KIP1) and MMP3 expression.