A De Novo Mouse Model of C11orf95-RELA Fusion-Driven Ependymoma Identifies Driver Functions in Addition to NF-κB.

The majority of supratentorial ependymomas (ST-ependymomas) have few mutations but frequently display chromothripsis of chromosome 11q that generates a fusion between C11orf95 and RELA (RELAFUS). Neural stem cells transduced with RELAFUSex vivo form ependymomas when implanted in the brain. These tumors display enhanced NF-κB signaling, suggesting that this aberrant signal is the principal mechanism of oncogenesis. However, it is not known whether RELAFUS is sufficient to drive de novo ependymoma tumorigenesis in the brain and, if so, whether these tumors also arise from neural stem cells. We show that RELAFUS drives ST-ependymoma formation from periventricular neural stem cells in mice and that RELAFUS-induced tumorigenesis is likely dependent on a series of cell signaling pathways in addition to NF-κB.

Corticosteroids compromise survival in glioblastoma.

Glioblastoma is the most common and most aggressive primary brain tumour. Standard of care consists of surgical resection followed by radiotherapy and concomitant and maintenance temozolomide (temozolomide/radiotherapy→temozolomide). Corticosteroids are commonly used perioperatively to control cerebral oedema and are frequently continued throughout subsequent treatment, notably radiotherapy, for amelioration of side effects. The effects of corticosteroids such as dexamethasone on cell growth in glioma models and on patient survival have remained controversial. We performed a retrospective analysis of glioblastoma patient cohorts to determine the prognostic role of steroid administration. A disease-relevant mouse model of glioblastoma was used to characterize the effects of dexamethasone on tumour cell proliferation and death, and to identify gene signatures associated with these effects. A murine anti-VEGFA antibody was used in parallel as an alternative for oedema control. We applied the dexamethasone-induced gene signature to The Cancer Genome Atlas glioblastoma dataset to explore the association of dexamethasone exposure with outcome. Mouse experiments were used to validate the effects of dexamethasone on survival in vivo Retrospective clinical analyses identified corticosteroid use during radiotherapy as an independent indicator of shorter survival in three independent patient cohorts. A dexamethasone-associated gene expression signature correlated with shorter survival in The Cancer Genome Atlas patient dataset. In glioma-bearing mice, dexamethasone pretreatment decreased tumour cell proliferation without affecting tumour cell viability, but reduced survival when combined with radiotherapy. Conversely, anti-VEGFA antibody decreased proliferation and increased tumour cell death, but did not affect survival when combined with radiotherapy. Clinical and mouse experimental data suggest that corticosteroids may decrease the effectiveness of treatment and shorten survival in glioblastoma. Dexamethasone-induced anti-proliferative effects may confer protection from radiotherapy- and chemotherapy-induced genotoxic stress. This study highlights the importance of identifying alternative agents such as vascular endothelial growth factor antagonists for managing oedema in glioblastoma patients. Beyond the established adverse effect profile of protracted corticosteroid use, this analysis substantiates the request for prudent and restricted use of corticosteroids in glioblastoma.

Synthesis and in vitro evaluation of novel 1,2,3,4-tetrahydroisoquinoline derivatives as potent antiglioma agents.

Glioblastoma Multiforme (GBM) continues to demand improved chemotherapeutic solutions. In order to discover novel chemotherapeutic agents for GBM, we identified novel tetrahydroisoquinoline (THI) analogs as antiglioma agents. The present study reports the design, synthesis and in vitro evaluation of new THI derivatives in four established human glioma cell lines (T98, U87, LN18 and A172). Our structure activity relationship (SAR) studies revealed that the important modification of the carbon linker between the biphenyl and THI ring yielded EDL-360 (12) as a potent antiglioma agent (LN18; IC50: 5.42 ± 0.06 µM) and is considered to be our new lead drug candidate for further preclinical studies.

EDL-360: A Potential Novel Antiglioma Agent.

Glioma is a brain tumor that arises from glial cells or glial progenitor cells, and represents 80% of malignant brain tumor incidence in the United States. Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor malignancy with fewer than 8% of patients with GBM surviving for more than 3 years. Over the past 10 years, despite improvement in diagnosis and therapies for cancer, the survival rate for high-grade glioma patients remains dismal. The main focus of our research is to identify potent novel antiglioma small molecules. We previously showed that EDL-360, a tetrahydroisoquinoline (THIQ) analog, as being highly cytotoxic to human glioma cell cultures. Here we show that EDL-360 significantly induced apoptosis in human glioma cell lines (U87 and LN18). However, in normal astrocytic cells, EDL-360 induced a modest G0/G1 cell cycle arrest but did not induce apoptosis. In an attempt to enhance EDL-360 induced cell death, we tested simultaneous treatment with EDL-360 and embelin (an inhibitor of the anti-apoptotic protein, XIAP). We found that, glioma cells had significant lower viability when EDL-360 and embelin were used in combination when compared to EDL-360 alone. We also used combination treatment of EDL-360 with decylubiquinone (dUb), a caspase-9 inhibitor, and found that the combination treatment induced a significant cell death when compared to treatment with EDL-360 alone. This is the first report that suggests that dUb has anticancer activity, and perhaps acts as a XIAP inhibitor. Finally, our in vivo data showed that EDL-360 treatment induced a partial regression in glioma tumorigenesis and induced cell death in the treated tumors as shown by H&E staining. Taken together these data suggests that EDL-360 has a potential therapeutic application for treating glioma, especially when combined with XIAP inhibitors.

Novel approaches to glioma drug design and drug screening.

INTRODUCTION: Gliomas are considered the most malignant form of brain tumors, and ranked among the most aggressive human cancers. Despite advance standard therapy the prognosis for patients with gliomas remains poor. Chemotherapy has played an important role as an adjuvant in treating gliomas. The efficacy of the chemotherapeutic drug is limited due to poor drug delivery and the inherent chemo- and radio-resistance. Challenges of the brain cancer therapy in clinical settings are; i) to overcome the chemo- and radio-resistance, ii) to improve drug delivery to tumors and iii) the development of effective drug screening procedures. AREAS COVERED: In this review, the authors discuss clinically important chemotherapeutic agents used for treating malignant gliomas along with novel drug design approaches. The authors, furthermore, discuss the in vitro and in vivo drug screening procedures for the development of novel drug candidates. EXPERT OPINION: The development of novel and highly potent chemotherapeutic agents for both glioma and glioma stem cells (GSCs) is highly important for future brain cancer research. Thus, research efforts should be directed towards developing innovative molecularly targeted antiglioma agents in order to reduce the toxicity and drug resistance which are associated with current forms of therapy. Development of novel pre-clinical drug screening procedures is also very critical for the overall success of brain cancer therapies in clinical settings.

PACSIN2 polymorphism influences TPMT activity and mercaptopurine-related gastrointestinal toxicity.

Treatment-related toxicity can be life-threatening and is the primary cause of interruption or discontinuation of chemotherapy for acute lymphoblastic leukemia (ALL), leading to an increased risk of relapse. Mercaptopurine is an essential component of continuation therapy in all ALL treatment protocols worldwide. Genetic polymorphisms in thiopurine S-methyltransferase (TPMT) are known to have a marked effect on mercaptopurine metabolism and toxicity; however, some patients with wild-type TPMT develop toxicity during mercaptopurine treatment for reasons that are not well understood. To identify additional genetic determinants of mercaptopurine toxicity, a genome-wide analysis was performed in a panel of human HapMap cell lines to identify trans-acting genes whose expression and/or single-nucleotide polymorphisms (SNPs) are related to TPMT activity, then validated in patients with ALL. The highest ranking gene with both mRNA expression and SNPs associated with TPMT activity in HapMap cell lines was protein kinase C and casein kinase substrate in neurons 2 (PACSIN2). The association of a PACSIN2 SNP (rs2413739) with TPMT activity was confirmed in patients and knock-down of PACSIN2 mRNA in human leukemia cells (NALM6) resulted in significantly lower TPMT activity. Moreover, this PACSIN2 SNP was significantly associated with the incidence of severe gastrointestinal (GI) toxicity during consolidation therapy containing mercaptopurine, and remained significant in a multivariate analysis including TPMT and SLCO1B1 as covariates, consistent with its influence on TPMT activity. The association with GI toxicity was also validated in a separate cohort of pediatric patients with ALL. These data indicate that polymorphism in PACSIN2 significantly modulates TPMT activity and influences the risk of GI toxicity associated with mercaptopurine therapy.

New substituted 4H-chromenes as anticancer agents.

As a continuation of our efforts to discover and develop small molecules as anticancer agents, we identified GRI-394837 as an initial hit from similarity search on RGD and its analogs. Based on GRI-394837, we designed and synthesized a focused set of novel chromenes (4a-e) in a single step using microwave method. All five compounds showed activity in the nanomolar range (IC(50): 7.4-640 nM) in two melanoma, three prostate and four glioma cancer cell lines. The chromene 4e is active against all the cell lines and particularly against the A172 human glioma cell line (IC(50): 7.4 nM). Interestingly, in vitro tubulin polymerization assay shows 4e to be a weak tubulin polymerization inhibitor but it shows very strong cytotoxicity in cellular assays, therefore there must be additional unknown mechanism(s) for the anticancer activity. Additionally, the strong antiproliferative activity was verified by one of the selected chromene (4a) by the NCI 60 cell line screen. These results strongly suggest that the novel chromenes could be further developed as a potential therapeutic agent for a variety of aggressive cancers.

Dantrolene sodium for the treatment of aldesleukin-induced rigors in a melanoma patient.

OBJECTIVE: To report the case of a 58-year-old male with melanoma who developed aldesleukin-induced rigors and was successfully treated with intravenous dantrolene sodium 20 mg and provide a review of the literature discussing other agents that have been used to treat drug-induced rigors. CASE SUMMARY: A 58-year-old male was treated with 720,000 IU/kg of aldesleukin every 8 hours as part of his antimelanoma therapy. The patient developed rigors after aldesleukin administration and was successfully treated with 25 mg of meperidine. Later, he experienced renal dysfunction that was also linked to aldesleukin therapy and developed normeperidine-induced neurotoxicity requiring discontinuation of meperidine therapy. The rigors were treated with intravenous dantrolene sodium 20 mg every 4 hours, with complete resolution of symptoms. DISCUSSION: Several antineoplastic agents can cause rigors; many of these agents can also lead to renal failure. Several agents have been investigated for their use in the management of rigors but can cause adverse effects or are unsuitable in the setting of renal insufficiency or failure. Although meperidine remains the mainstay for the treatment and prevention of rigors, it can be associated with neurotoxicity in some patients, particularly those with impaired renal function. Given that dantrolene has been shown to be effective against rigors, it may be a useful alternative for patients who can not tolerate meperidine. Drugs with a more favorable adverse effect profile that are not eliminated through the kidneys are needed. CONCLUSIONS: In the oncology setting, severe rigors can result in the interruption of a patient's cancer therapy, which can increase the risk of treatment failure. Dantrolene may be a useful alternative for patients experiencing rigors who can not tolerate meperidine.

Animal models for glioma drug discovery.

INTRODUCTION: High-grade gliomas are among the most deadly of all cancer types and are also the most common malignant primary tumors of the CNS. Large-scale studies that have analyzed the transcriptional and translational expression patterns of glioma have found that the majority of these tumors can be categorized based on specific genomic anomalies. Genetically engineered mouse models (GEMMs) that represent the molecular subgroups of the human disease harbor a variety of molecular alterations that have been proven to drive gliomagenesis. These models provide an opportunity to assess the effects of novel therapies in the presence of specific molecular defects. Research using GEMMs, which are associated with these subclasses, allow researchers to assess drug efficacy by subclass. AREAS COVERED: In this review, the authors discuss the histological and molecular characteristics of malignant gliomas, the therapies used to treat them and the animal models that closely recapitulate them. EXPERT OPINION: It is likely that GEMMs that recapitulate the molecular character of human tumors will provide a more accurate prediction of individuals who may be more or less likely to benefit from specific therapies. This knowledge can be then used to drive clinical trial design and this, in turn, could lead to better therapeutic outcomes.

Molecular pathogenesis of malignant glial tumors.

Malignant glial tumors are the most aggressive and difficult to treat neoplasms arising in the brain. More than 22,000 people in the United States are diagnosed with a malignant glioma annually, and most will die within the first two years from diagnosis. Traditionally, gliomas have been categorized based solely on tumor histological features. However, expression studies have found that molecular signatures can be used to categorize these tumors into subclasses that more effectively predict patient outcome. The heterogeneity between tumors as well as within individual tumors makes understanding the molecular aspects of tumorigenesis extremely important. Several genetically engineered mouse models (GEMMs) of glioma have been developed that recapitulate the molecular alterations observed in the human disease. GEMMs of glioma have allowed researchers to more closely study the role of cancer stem cells (CSC) in gliomagenesis as well as the relevance of signaling within the CSC microenvironment. Knowledge of the underlying molecular signatures of malignant glial tumors coupled with the existence of a variety of human disease-relevant GEMMs of this tumor type provide researchers and clinicians with valuable resources for the discovery of new drug targets.

Thiopurine methyltransferase predicts the extent of cytotoxicty and DNA damage in astroglial cells after thioguanine exposure.

Thiopurine methyltransferase (Tpmt) is the primary enzyme responsible for deactivating thiopurine drugs. Thiopurine drugs (i.e., thioguanine [TG], mercaptopurine, azathioprine) are commonly used for the treatment of cancer, organ transplant, and autoimmune disorders. Chronic thiopurine therapy has been linked to the development of brain cancer (most commonly astrocytomas), and Tpmt status has been associated with this risk. Therefore, we investigated whether the level of Tpmt protein activity could predict TG-associated cytotoxicity and DNA damage in astrocytic cells. We found that TG induced cytotoxicity in a dose-dependent manner in Tpmt(+/+), Tpmt(+/-) and Tpmt(-/-) primary mouse astrocytes and that a low Tpmt phenotype predicted significantly higher sensitivity to TG than did a high Tpmt phenotype. We also found that TG exposure induced significantly more DNA damage in the form of single strand breaks (SSBs) and double strand breaks (DSBs) in primary astrocytes with low Tpmt versus high Tpmt. More interestingly, we found that Tpmt(+/-) astrocytes had the highest degree of cytotoxicity and genotoxicity (i.e., IC(50), SSBs and DSBs) after TG exposure. We then used human glioma cell lines as model astroglial cells to represent high (T98) and low (A172) Tpmt expressers and found that A172 had the highest degree of cytoxicity and SSBs after TG exposure. When we over-expressed Tpmt in the A172 cell line, we found that TG IC(50) was significantly higher and SSB's were significantly lower as compared to mock transfected cells. This study shows that low Tpmt can lead to greater sensitivity to thiopurine therapy in astroglial cells. When Tpmt deactivation at the germ-line is considered, this study also suggests that heterozygosity may be subject to the greatest genotoxic effects of thiopurine therapy.

Delivery of temozolomide to the tumor bed via biodegradable gel matrices in a novel model of intracranial glioma with resection.

INTRODUCTION: We have completed in vivo safety and efficacy studies of the use of a novel drug delivery system, a gel matrix-temozolomide formulation that is injected intracranially into the post-resection cavity, as a candidate for glioma therapy. METHODS: A rat intracranial resection model of C6-GFP intracranial glioma was used for safety and toxicity studies. Biodistribution studies were performed using gel matrix-gallocyanine formulations and were evaluated at various time intervals using real-time analysis of dye distribution. Additionally, the resection model was used to determine the efficacy of gel matrix-temozolomide as compared to blank gel matrix. A subcutaneous human xenograft glioma model was used to further assess the efficacy of gel matrix-temozolomide in reducing the overall tumor load. RESULTS: Gel matrix-temozolomide exhibited minimal cytotoxicity toward normal brain tissue while displaying high levels of oncolytic activity toward glioma cells. In the intracranial glioma resection and subcutaneous glioma model, administration of gel matrix-temozolomide directly to the tumor bed was well tolerated and effective at reducing the tumor load. A significant reduction of tumor load was observed (P < 0.0001) in the 30% temozolomide group (approximately 95%) as compared to blank control. There was little morbidity and no mortality associated with gel matrix treatment. CONCLUSIONS: Gel matrix-temozolomide appears to be safe and effective when used in vivo to treat intracranial glioma and warrants further development as a potential adjuvant therapy.

CRHR1 polymorphisms predict bone density in survivors of acute lymphoblastic leukemia.

PURPOSE: Corticosteroids are a critical component of therapy for acute lymphoblastic leukemia (ALL) but are associated with late effects, such as osteoporosis. Risk factors remain poorly defined. Because CRHR1 polymorphisms have been associated with other corticosteroid effects, our goal was to define whether CRHR1 polymorphisms predict which patients with ALL are likely to develop bone mineral deficits. PATIENTS AND METHODS: The mean bone mineral density z scores of 309 long-term survivors of ALL were determined by quantitative computed tomography of the trabecular lumbar spine. We analyzed whether CRHR1 genotypes, adjusted for sex, ALL treatment regimen, and weight, could predict bone density. RESULTS: We found that three single nucleotide polymorphisms (SNPs), all in linkage disequilibrium, were associated with bone density in a sex-specific manner. Bone density was lower in males (P = .001), in nonblack patients (P < .08), in those who were not overweight (P < .001), and in those who received intensive antimetabolites and glucocorticoids (P < .001). After adjustment for these features, the G allele at the rs1876828 SNP was associated with lower z scores (P = .02) in males but tended to have the opposite association in females (P = .09). CONCLUSION: CRHR1 polymorphisms may impact the risk of bone density deficits in patients treated with corticosteroids and antimetabolites in a sex-specific manner.

Safety and efficacy of a novel cannabinoid chemotherapeutic, KM-233, for the treatment of high-grade glioma.

OBJECTIVE: To test in vitro and in vivo the safety and efficacy of a novel chemotherapeutic agent, KM-233, for the treatment of glioma. METHODS: In vitro cell cytotoxicity assays were used to measure and compare the cytotoxic effects of KM-233, Delta(8)-tetrahydrocannabinol (THC), and bis-chloroethyl-nitrosurea (BCNU) against human U87 glioma cells. An organotypic brain slice culture model was used for safety and toxicity studies. A human glioma-SCID mouse side-pocket tumor model was used to test in vivo the safety and efficacy of KM-233 with intratumoral and intra-peritoneal administration. RESULTS: KM-233 is a classical cannabinoid with good blood brain barrier penetration that possesses a selective affinity for the CB2 receptors relative to THC. KM-233 was as efficacious in its cytotoxicity against human U87 glioma as Delta(8)-tetrahydrocannabinol, and superior to the commonly used anti-glioma chemotherapeutic agent, BCNU. The cytotoxic effects of KM-233 against human glioma cells in vitro occur as early as two hours after administration, and dosing of KM-233 can be cycled without compromising cytotoxic efficacy and while improving safety. Cyclical dosing of KM-233 to treat U87 glioma in a SCID mouse xenograft side pocket model was effective at reducing the tumor burden with both systemic and intratumoral administration. CONCLUSION: These studies provide both in vitro and in vivo evidence that KM-233 shows promising efficacy against human glioma cell lines in both in vitro and in vivo studies, minimal toxicity to healthy cultured brain tissue, and should be considered for definitive preclinical development in animal models of glioma.