Activation of the Mevalonate Pathway in Response to Anti-cancer Treatments Drives Glioblastoma Recurrences Through Activation of Rac-1.

Glioblastoma (GBM) is the deadliest adult brain cancer. Under the current standard of care, almost all patients succumb to the disease and novel treatments are urgently needed. Recognizing that GBMs are addicted to cholesterol, past clinical trials have repurposed statins against GBM but failed. The purpose of this study was to test whether treatments that upregulate the cholesterol biosynthesis pathway in GBM would generate a metabolic vulnerability that can be exploited using statins and to determine the underlying mechanisms.Effects of radiotherapy and temozolomide or dopamine receptor antagonists on the mevalonate pathway in GBM were assessed in vitro and in vivo. The impact of statins on self-renewal of glioma stem cells and median survival was studied. Branches of the mevalonate pathway were probed to identify relevant effector proteins.Cells surviving combination treatments that converge in activating the immediate early response, universally upregulated the mevalonate pathway and increased stemness of GBM cells through activation of the Rho-GTPase Rac-1. Activation of the mevalonate pathway and Rac-1 was inhibited by statins, which led to improved survival in mouse models of glioblastoma when combined with radiation and drugs that target the glioma stem cell pool and plasticity of glioma cells.We conclude that a combination of dopamine receptor antagonists and statins could potentially improve radiotherapy outcome and warrants further investigation. SIGNIFICANCE: Combination therapies that activate the mevalonate pathway in GBM cells after sublethal treatment enhance self-renewal and migratory capacity through Rac-1 activation, which creates a metabolic vulnerability that can be further potentially exploited using statins.

Radiation-Induced Cellular Plasticity: A Strategy for Combatting Glioblastoma.

Glioblastoma is the deadliest brain cancer in adults and almost all patients succumb to the tumor. While surgery followed by chemo-radiotherapy significantly delays disease progression, these treatments do not lead to long-term tumor control and targeted therapies or biologics have so far failed to further improve survival. Utilizing a transient radiation-induced state of multipotency we used the adenylcyclase activator forskolin to alter the cellular fate of glioma cells in response to radiation. The combined treatment induced the expression of neuronal markers in glioma cells, reduced proliferation and led to a distinct gene expression profile. scRNAseq revealed that the combined treatment forced glioma cells into a microglia- and neuron-like phenotypes. In vivo this treatment led to a loss of glioma stem cells and prolonged median survival in mouse models of glioblastoma. Collectively, our data suggest that revisiting a differentiation therapy with forskolin in combination with radiation could lead to clinical benefit.

Activation of the mevalonate pathway in response to anti-cancer treatments drives glioblastoma recurrences through activation of Rac-1.

Glioblastoma is the deadliest adult brain cancer. Under the current standard of care almost all patients succumb to the disease and novel treatments are urgently needed. Dopamine receptor antagonists have been shown to target cancer cell plasticity in GBM and repurposing these FDA-approved drugs in combination with radiation improves the efficacy of radiotherapy in glioma models. In cells surviving this combination treatment the mevalonate pathway is upregulated at the transcriptional and functional level. Here we report that glioblastoma treatments that converge in the immediate early response to radiation through activation of the MAPK cascade universally upregulate the mevalonate pathway and increase stemness of GBM cells through activation of the Rho-GTPase Rac-1. Activation of the mevalonate pathway and Rac-1 is inhibited by statins, which leads to improved survival in mouse models of glioblastoma when combined with radiation and drugs that target the glioma stem cell pool and plasticity of glioma cells.

Effects of Dopamine Receptor Antagonists and Radiation on Mouse Neural Stem/Progenitor Cells.

Background: Dopamine receptor antagonists are psychotropic drugs that have been originally developed against psychiatric disorders. We recently identified dopamine receptor antagonists as potential anti-cancer agents and some have entered clinical trials against glioblastoma. Radiotherapy is known to cause cognitive impairment in patients receiving cranial irradiation through the elimination of neural stem/progenitor cells and subsequent loss of neurogenesis. Methods: Using transgenic mice that report the presence of neural stem/progenitor cells through Nestin promoter-driven expression of enhanced green fluorescent protein, the effects of dopamine receptor antagonists alone or in combination with radiation on murine neural stem/progenitor cells were assessed in sphere-formation assays, flow cytometry and immunofluorescence in vitro and in vivo . Results: We report that several dopamine receptor antagonists show sex-dependent effects on neural stem/progenitor cells both in vitro and in vivo . Hydroxyzine, trifluoperazine, amisulpride, nemonapride or quetiapine alone or in combination with radiation significantly increased the number of neural stem/progenitor cells in female neurospheres but not in male mice. Dopamine receptor antagonists either protected neural stem/progenitor cells from radiation or expanded the stem cell pool, thus indicating that this combination therapy against glioblastoma will not increase radiation-induced cognitive decline through increasing elimination of neural stem/progenitor cells and subsequent loss of neurogenesis. Conclusions: We conclude that a therapeutic window for dopamine receptor antagonists in combination with radiation potentially exist, making it a novel combination therapy against glioblastoma. Normal tissue toxicity of this combination potentially differs depending on age and sex and should be taken into consideration when designing clinical trials. Key Points: - Neural stem/progenitor cells show sex-dependent sensitivity to dopamine receptor antagonists- Dopamine receptor antagonists active against GBM increase Neural stem/progenitor cells counts. Importance of the Study: Combination therapy of dopamine receptor antagonists with radiation have entered clinical trials against glioblastoma but the normal tissue toxicity of this combination has not been fully explored yet. Here we present evidence that some dopamine receptor antagonists show sex-dependent effects on neural stem/progenitor cells either by protecting neural stem/progenitor cells from radiation or inducing an expansion of the stem cell pool, suggesting that this combination therapy against glioblastoma will not increase radiation-induced cognitive decline through increasing elimination of neural stem/progenitor cells and subsequent loss of neurogenesis. Normal tissue toxicity of this combination potentially differs depending on age and sex and should be further explored in clinical trials.

Trichothiodystrophy hair shafts display distinct ultrastructural features.

Hair shafts from three trichothiodystrophy (TTD) patients with mutations in the ERCC2 (XPD) gene were examined by transmission electron microscopy. TTD is a rare, recessive disorder with mutations in several genes in the DNA repair/transcription pathway, including ERCC2. Unlike previous studies, the hair shafts were examined after relaxation of their structure by partial disulphide bond reduction in the presence of sodium dodecyl sulphate, permitting improved visualization. Compared with hair shafts of normal phenotype, TTD cuticle cells displayed aberrant marginal bands and exocuticle layers. Clusters of cells stained differently (light versus dark) in the cortex of aberrant shafts, and the keratin macrofibrils appeared much shorter in the cytoplasm. Considerable heterogeneity in these properties was evident among samples and even along the length of single hair shafts. The results are consistent with not only a paucity of high sulphur components, such as keratin-associated proteins, but also a profound imbalance in protein content and organization.

Effects of the DRD2/3 antagonist ONC201 and radiation in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the deadliest of all brain cancers in adults. The current standard-of-care is surgery followed by radiotherapy and temozolomide, leading to a median survival time of only 15 months. GBM are organized hierarchically with a small number of glioma-initiating cells (GICs), responsible for therapy resistance and tumor recurrence, suggesting that targeting GICs could improve treatment response. ONC201 is a first-in-class anti-tumor agent with clinical efficacy in some forms of high-grade gliomas. Here we test its efficacy against GBM in combination with radiation. METHODS: Using patient-derived GBM lines and mouse models of GBM we test the effects of radiation and ONC201 on GBM self-renewalin vitro and survivalin vivo.A possible resistance mechanism is investigated using RNA-Sequencing. RESULTS: Treatment of GBM cells with ONC201 reduced self-renewal, clonogenicity and cell viabilityin vitro. ONC201 exhibited anti-tumor effects on radioresistant GBM cells indicated by reduced self-renewal in secondary and tertiary glioma spheres. Combined treatment of ONC201 and radiation prolonged survival in syngeneic and patient-derived orthotopic xenograft mouse models of GBM. Subsequent transcriptome analyses after combined treatment revealed shifts in gene expression signatures related to quiescent GBM populations, GBM plasticity, and GBM stem cells. CONCLUSIONS: Our findings suggest that combined treatment with the DRD2/3 antagonist ONC201 and radiation improves the efficacy of radiation against GBMin vitroandin vivothrough suppression of GICs without increasing toxicity in mouse models of GBM. A clinical assessment of this novel combination therapy against GBM is further warranted.

Dopamine Receptor Antagonists, Radiation, and Cholesterol Biosynthesis in Mouse Models of Glioblastoma.

BACKGROUND: Glioblastoma is the deadliest brain tumor in adults, and the standard of care consists of surgery followed by radiation and treatment with temozolomide. Overall survival times for patients suffering from glioblastoma are unacceptably low indicating an unmet need for novel treatment options. METHODS: Using patient-derived HK-157, HK-308, HK-374, and HK-382 glioblastoma lines, the GL261 orthotopic mouse models of glioblastoma, and HK-374 patient-derived orthotopic xenografts, we tested the effect of radiation and the dopamine receptor antagonist quetiapine on glioblastoma self-renewal in vitro and survival in vivo. A possible resistance mechanism was investigated using RNA-sequencing. The blood-brain-barrier-penetrating statin atorvastatin was used to overcome this resistance mechanism. All statistical tests were 2-sided. RESULTS: Treatment of glioma cells with the dopamine receptor antagonist quetiapine reduced glioma cell self-renewal in vitro, and combined treatment of mice with quetiapine and radiation prolonged the survival of glioma-bearing mice. The combined treatment induced the expression of genes involved in cholesterol biosynthesis. This rendered GL261 and HK-374 orthotopic tumors vulnerable to simultaneous treatment with atorvastatin and further statistically significantly prolonged the survival of C57BL/6 (n = 10 to 16 mice per group; median survival not reached; log-rank test, P < .001) and NOD Scid gamma mice (n = 8 to 21 mice per group; hazard ratio = 3.96, 95% confidence interval = 0.29 to 12.40; log-rank test, P < .001), respectively. CONCLUSIONS: Our results indicate promising therapeutic efficacy with the triple combination of quetiapine, atorvastatin, and radiation treatment against glioblastoma without increasing the toxicity of radiation. With both drugs readily available for clinical use, our study could be rapidly translated into a clinical trial.

Tumor necrosis factor receptor signaling modulates carcinogenesis in a mouse model of breast cancer.

Pro-inflammatory conditions have long been associated with mammary carcinogenesis and breast cancer progression. The underlying mechanisms are incompletely understood but signaling of pro-inflammatory cytokine TNFα through its receptors TNFR1 and TNFR2 is a major mediator of inflammation in both obesity and in the response of tissues to radiation, 2 known risk factors for the development of breast cancer. Here, we demonstrated the loss of one TNFR2 allele led to ductal hyperplasia in the mammary gland with increased numbers of mammary epithelial stem cell and terminal end buds. Furthermore, loss of one TNFR2 allele increased the incidence of breast cancer in MMTV-Wnt1 mice and resulted in tumors with a more aggressive phenotype and metastatic potential. The underlying mechanisms include a preferential activation of canonical NF-κB signaling pathway and autocrine production of TNFα. Analysis of the TCGA dataset indicated inferior overall survival for patients with down-regulated TNFR2 expression. These findings unravel the imbalances in TNFR signaling promote the development and progression of breast cancer, indicating that selective agonists of TNFR2 could potentially modulate the risk for breast cancer in high-risk populations.

The worldwide clinical trial research response to the COVID-19 pandemic - the first 100 days.

Background: Never before have clinical trials drawn as much public attention as those testing interventions for COVID-19. We aimed to describe the worldwide COVID-19 clinical research response and its evolution over the first 100 days of the pandemic. Methods: Descriptive analysis of planned, ongoing or completed trials by April 9, 2020 testing any intervention to treat or prevent COVID-19, systematically identified in trial registries, preprint servers, and literature databases. A survey was conducted of all trials to assess their recruitment status up to July 6, 2020. Results: Most of the 689 trials (overall target sample size 396,366) were small (median sample size 120; interquartile range [IQR] 60-300) but randomized (75.8%; n=522) and were often conducted in China (51.1%; n=352) or the USA (11%; n=76). 525 trials (76.2%) planned to include 155,571 hospitalized patients, and 25 (3.6%) planned to include 96,821 health-care workers. Treatments were evaluated in 607 trials (88.1%), frequently antivirals (n=144) or antimalarials (n=112); 78 trials (11.3%) focused on prevention, including 14 vaccine trials. No trial investigated social distancing. Interventions tested in 11 trials with >5,000 participants were also tested in 169 smaller trials (median sample size 273; IQR 90-700). Hydroxychloroquine alone was investigated in 110 trials. While 414 trials (60.0%) expected completion in 2020, only 35 trials (4.1%; 3,071 participants) were completed by July 6. Of 112 trials with detailed recruitment information, 55 had recruited <20% of the targeted sample; 27 between 20-50%; and 30 over 50% (median 14.8% [IQR 2.0-62.0%]). Conclusions: The size and speed of the COVID-19 clinical trials agenda is unprecedented. However, most trials were small investigating a small fraction of treatment options. The feasibility of this research agenda is questionable, and many trials may end in futility, wasting research resources. Much better coordination is needed to respond to global health threats.

1-[(4-Nitrophenyl)sulfonyl]-4-phenylpiperazine treatment after brain irradiation preserves cognitive function in mice.

BACKGROUND: Normal tissue toxicity is an inevitable consequence of primary or secondary brain tumor radiotherapy. Cranial irradiation commonly leads to neurocognitive deficits that manifest months or years after treatment. Mechanistically, radiation-induced loss of neural stem/progenitor cells, neuroinflammation, and demyelination are contributing factors that lead to progressive cognitive decline. METHODS: The effects of 1-[(4-nitrophenyl)sulfonyl]-4-phenylpiperazine (NSPP) on irradiated murine neurospheres, microglia cells, and patient-derived gliomaspheres were assessed by sphere-formation assays, flow cytometry, and interleukin (IL)-6 enzyme-linked immunosorbent assay. Activation of the hedgehog pathway was studied by quantitative reverse transcription PCR. The in vivo effects of NSPP were analyzed using flow cytometry, sphere-formation assays, immunohistochemistry, behavioral testing, and an intracranial mouse model of glioblastoma. RESULTS: We report that NSPP mitigates radiation-induced normal tissue toxicity in the brains of mice. NSPP treatment significantly increased the number of neural stem/progenitor cells after brain irradiation in female animals, and inhibited radiation-induced microglia activation and expression of the pro-inflammatory cytokine IL-6. Behavioral testing revealed that treatment with NSPP after radiotherapy was able to successfully mitigate radiation-induced decline in memory function of the brain. In mouse models of glioblastoma, NSPP showed no toxicity and did not interfere with the growth-delaying effects of radiation. CONCLUSIONS: We conclude that NSPP has the potential to mitigate cognitive decline in patients undergoing partial or whole brain irradiation without promoting tumor growth and that the use of this compound as a radiation mitigator of radiation late effects on the central nervous system warrants further investigation.