Diffuse white matter injury (DWMI), a leading cause of neurodevelopmental disabilities in preterm infants, is characterized by reduced oligodendrocyte formation. NG2-expressing oligodendrocyte ...precursor cells (NG2 cells) are exposed to various extrinsic regulatory signals, including the neurotransmitter GABA. We investigated GABAergic signaling to cerebellar white matter NG2 cells in a mouse model of DWMI (chronic neonatal hypoxia). We found that hypoxia caused a loss of GABAA receptor-mediated synaptic input to NG2 cells, extensive proliferation of these cells and delayed oligodendrocyte maturation, leading to dysmyelination. Treatment of control mice with a GABAA receptor antagonist or deletion of the chloride-accumulating transporter NKCC1 mimicked the effects of hypoxia. Conversely, blockade of GABA catabolism or GABA uptake reduced NG2 cell numbers and increased the formation of mature oligodendrocytes both in control and hypoxic mice. Our results indicate that GABAergic signaling regulates NG2 cell differentiation and proliferation in vivo, and suggest that its perturbation is a key factor in DWMI.
Purpose: Telomerase activity is one of the hallmarks of cancer and is a highly relevant therapeutic target. The effects of a novel
human telomerase antagonist, imetelstat, on primary human ...glioblastoma (GBM) tumor-initiating cells were investigated in vitro and in vivo .
Experimental Design: Tumor-initiating cells were isolated from primary GBM tumors and expanded as neurospheres in vitro . The GBM tumor-initiating cells were treated with imetelstat and examined for the effects on telomerase activity levels,
telomere length, proliferation, clonogenicity, and differentiation. Subsequently, mouse orthotopic and subcutaneous xenografts
were used to assess the in vivo efficacy of imetelstat.
Results: Imetelstat treatment produced a dose-dependent inhibition of telomerase (IC 50 0.45 μmol/L). Long-term imetelstat treatment led to progressive telomere shortening, reduced rates of proliferation, and
eventually cell death in GBM tumor-initiating cells. Imetelstat in combination with radiation and temozolomide had a dramatic
effect on cell survival and activated the DNA damage response pathway. Imetelstat is able to cross the blood-brain barrier
in orthotopic GBM xenograft tumors. Fluorescently labeled GBM tumor cells isolated from orthotopic tumors, following systemic
administration of imetelstat (30 mg/kg every day for three days), showed ∼70% inhibition of telomerase activity. Chronic systemic
treatment produced a marked decrease in the rate of xenograft subcutaneous tumor growth.
Conclusion: This preclinical study supports the feasibility of testing imetelstat in the treatment of GBM patients, alone or in combination
with standard therapies. Clin Cancer Res; 16(1); 154–63
Glioblastoma multiforme (GBM) is the most lethal of brain tumors and is highly resistant to ionizing radiation (IR) and chemotherapy. Here, we report on a molecular mechanism by which a key ...glioma-specific mutation, epidermal growth factor receptor variant III (EGFRvIII), confers radiation resistance. Using Ink4a/Arf-deficient primary mouse astrocytes, primary astrocytes immortalized by p53/Rb suppression, as well as human U87 glioma cells, we show that EGFRvIII expression enhances clonogenic survival following IR. This enhanced radioresistance is due to accelerated repair of DNA double-strand breaks (DSB), the most lethal lesion inflicted by IR. The EGFR inhibitor gefitinib (Iressa) and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 attenuate the rate of DSB repair. Importantly, expression of constitutively active, myristylated Akt-1 accelerates repair, implicating the PI3K/Akt-1 pathway in radioresistance. Most notably, EGFRvIII-expressing U87 glioma cells show elevated activation of a key DSB repair enzyme, DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Enhanced radioresistance is abrogated by the DNA-PKcs-specific inhibitor NU7026, and EGFRvIII fails to confer radioresistance in DNA-PKcs-deficient cells. In vivo, orthotopic U87-EGFRvIII-derived tumors display faster rates of DSB repair following whole-brain radiotherapy compared with U87-derived tumors. Consequently, EGFRvIII-expressing tumors are radioresistant and continue to grow following whole-brain radiotherapy with little effect on overall survival. These in vitro and in vivo data support our hypothesis that EGFRvIII expression promotes DNA-PKcs activation and DSB repair, perhaps as a consequence of hyperactivated PI3K/Akt-1 signaling. Taken together, our results raise the possibility that EGFR and/or DNA-PKcs inhibition concurrent with radiation may be an effective therapeutic strategy for radiosensitizing high-grade gliomas.
Human papillomavirus (HPV) positive and negative head and neck squamous cell carcinoma (HNSCC) are known to have differential phenotypes, including the incidence and location of metastases. HPV ...positive (HPV+) HNSCC are more likely to metastasize to distant sites, such as the lung, brain, and skin. Among these locations, metastasis to the brain is a rare event, and little is known about specific risk factors for this phenotype. In this report, we describe two patients who developed brain metastases from HNSCC. Both patient tumors had p16
overexpression, suggesting these tumors were HPV+. This was confirmed after PCR, in situ hybridization, and mass spectrometry detected the presence of HPV type 16 (HPV16) DNA, RNA and protein. To further characterize the presence of HPV16, we used a target enrichment strategy on tumor DNA and RNA to isolate the viral sequences from the brain metastases. Analysis by targeted next generation sequencing revealed that both tumors had the HPV genome integrated into the host genome at known hotspots, 8q24.21 and 14q24.1. Applying a similar target enrichment strategy to a larger cohort of HPV+ HNSCC brain metastases could help to identify biomarkers that can predict metastasis and/or identify novel therapeutic options.
Reactive astrogliosis is an essential and ubiquitous response to CNS injury, but in some cases, aberrant activation of astrocytes and their release of inhibitory signaling molecules can impair ...endogenous neural repair processes. Our lab previously identified a secreted intercellular signaling molecule, called endothelin-1 (ET-1), which is expressed at high levels by reactive astrocytes in multiple sclerosis (MS) lesions and limits repair by delaying oligodendrocyte progenitor cell (OPC) maturation. However, as ET receptors are widely expressed on neural cells, the cell- and receptor-specific mechanisms of OPC inhibition by ET-1 action remain undefined. Using pharmacological approaches and cell-specific endothelin receptor (EDNR) ablation, we show that ET-1 acts selectively through EDNRB on astrocytes—and not OPCs—to indirectly inhibit remyelination. These results demonstrate that targeting specific pathways in reactive astrocytes represents a promising therapeutic target in diseases with extensive reactive astrogliosis, including MS.
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•EDNRA and EDNRB are upregulated after demyelination in reactive astrocytes•Pharmacological inhibition of EDNRB, but not EDNRA, accelerates remyelination•EDNRB loss in astrocytes, but not in OPCs, accelerates remyelination•Endothelin indirectly inhibits OPC differentiation through astrocytes
Astrocyte-derived endothelin-1 (ET-1) inhibits remyelination through unknown mechanisms. Using pharmacological and genetic approaches, Hammond et al. demonstrate that ET-1 signals through endothelin receptor-B in reactive astrocytes, indirectly inhibiting oligodendrocyte progenitor cell (OPC) differentiation and remyelination. Inhibiting this pathway could provide an exciting therapeutic strategy to promote remyelination in MS.
Sox17, a SoxF family member transiently upregulated during postnatal oligodendrocyte (OL) development, promotes OL cell differentiation, but its function in white matter development and pathology ...in vivo is unknown. Our analysis of oligodendroglial- and OL-progenitor-cell-targeted ablation in vivo using a floxed Sox17 mouse establishes a dependence of postnatal oligodendrogenesis on Sox17 and reveals Notch signaling as a mediator of Sox17 function. Following Sox17 ablation, reduced numbers of Olig2-expressing cells and mature OLs led to developmental hypomyelination and motor dysfunction. After demyelination, Sox17 deficiency inhibited OL regeneration. OL decline was unexpectedly preceded by transiently increased differentiation and a reduction of OL progenitor cells. Evidence of a dual role for Sox17 in progenitor cell expansion by Notch and differentiation involving TCF7L2 expression were found. A program of progenitor expansion and differentiation promoted by Sox17 through Notch thus contributes to OL production and determines the outcome of white matter repair.
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•Sox17 ablation in CNS in vivo impairs oligodendrocyte development and regeneration•Sox17 promotes both oligodendrocyte progenitor expansion and maturation in vivo•Notch signaling mediates oligodendrocyte progenitor cell maintenance by Sox17•TCF7L2 expression in oligodendrocytes is regulated by Sox17 and Notch signaling
Oligodendrocyte development is controlled by sequential processes of progenitor cell proliferation and differentiation, believed to be largely regulated by distinct mechanisms. Chew et al. characterize white matter abnormalities in brains of Sox17-deficient mice and identify the candidate Sox17-regulated target genes Notch and TCF7L2, which mediate progenitor cell expansion and maturation, respectively.
Glioblastomas (GBM) are lethal brain tumors that are highly resistant to therapy. The only meaningful improvement in therapeutic response came from use of the S(N)1-type alkylating agent temozolomide ...in combination with ionizing radiation. However, no genetic markers that might predict a better response to DNA alkylating agents have been identified in GBMs, except for loss of O(6-)methylguanine-DNA methyltransferase via promoter methylation. In this study, using genetically defined primary murine astrocytes as well as human glioma lines, we show that loss of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) confers sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a functional analogue of temozolomide. We find that MNNG induces replication-associated DNA double-strand breaks (DSB), which are inefficiently repaired in PTEN-deficient astrocytes and trigger apoptosis. Mechanistically, this is because PTEN-null astrocytes are compromised in homologous recombination (HR), which is important for the repair of replication-associated DSBs. Our results suggest that reduced levels of Rad51 paralogs in PTEN-null astrocytes might underlie the HR deficiency of these cells. Importantly, the HR deficiency of PTEN-null cells renders them sensitive to the poly(ADP-ribose) polymerase (PARP) inhibitor ABT-888 due to synthetic lethality. In sum, our results tentatively suggest that patients with PTEN-null GBMs (about 36%) may especially benefit from treatment with DNA alkylating agents such as temozolomide. Significantly, our results also provide a rational basis for treating the subgroup of patients who are PTEN deficient with PARP inhibitors in addition to the current treatment regimen of radiation and temozolomide.
Abstract Background: The human genome encodes more than 1,600 transcription factors (TFs), along with additional cofactors, chromatin regulators, and structural proteins that collectively execute the ...regulatory instructions encoded within the nuclear DNA. Dysfunctions of these proteins, collectively known as the Regulome, are known to drive multiple conditions such as cancer, autoimmune conditions, and fibrosis. Despite their importance, many of these proteins are considered undruggable due to challenges in modeling their activity in vitro. Methods: We have addressed these shortcomings by implementing a live-cell Regulome profiling approach that quantifies the global activity of TFs without the need for cell manipulation or genome engineering, called TF-Scan. The technology coupled rapid, automated purification of the DNA-bound proteome coupled to scalable Data Independent Acquisition (DIA) quantitative mass spectrometry proteomics and machine learning. Results: We completed a screen of covalent compounds across 8 tissue types to discover hotspot sites on TFs that control their DNA-binding activity. A chordoma cell line was included in this screen. The screen identified compounds capable of binding to and disrupting the activity of several previously undruggable transcription factors, including brachyury. Hit validation and hit-to-lead medicinal chemistry resulted in a covalent chemical probe for brachyury, with activity in cell line models of chordoma. Conclusion: Phenotypic drug discovery in live, unmodified human cells with systematic regulome profiling enables the discovery of compounds that disrupt previously undruggable targets by multiple mechanisms of action. Citation Format: Alexander Joel Federation, Yang Gao, Brian McEllin, Tonibelle Gatbonton-Schwager, Andrea Gutierrez, Julia Robbins, Bodhi Hueffmeier, Erin Broderick, Daniele Canzani, William Fondrie, Lindsay Pino. Profiling the DNA regulome to discover direct inhibitors of the brachyury transcription factor abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3225.
Glioblastomas (GBM) are highly radioresistant and lethal brain tumors. Ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) are a risk factor for the development of GBM. In this study, we ...systematically examined the contribution of IR-induced DSBs to GBM development using transgenic mouse models harboring brain-targeted deletions of key tumor suppressors frequently lost in GBM, namely Ink4a, Ink4b, Arf and/or PTEN. Using low linear energy transfer (LET) X-rays to generate simple breaks or high LET HZE particles (Fe ions) to generate complex breaks, we found that DSBs induce high-grade gliomas in these mice which, otherwise, do not develop gliomas spontaneously. Loss of Ink4a and Arf was sufficient to trigger IR-induced glioma development but additional loss of Ink4b significantly increased tumor incidence. We analyzed IR-induced tumors for copy number alterations to identify oncogenic changes that were generated and selected for as a consequence of stochastic DSB events. We found Met amplification to be the most significant oncogenic event in these radiation-induced gliomas. Importantly, Met activation resulted in the expression of Sox2, a GBM cancer stem cell marker, and was obligatory for tumor formation. In sum, these results indicate that radiation-induced DSBs cooperate with loss of Ink4 and Arf tumor suppressors to generate high-grade gliomas that are commonly driven by Met amplification and activation.
Abstract
Glioblastoma multiforme (GBM) are highly lethal brain tumors for which exposure to ionizing radiation is the only known risk factor. GBMs are characterized by alterations in three core ...signaling pathways: 1) RTK-PI3K-Akt, 2) ARF-MDM2-p53, and 3) Ink4a-RB1. In order to objectively evaluate the risk of developing malignant gliomas from exposure to ionizing radiation, we have developed mouse models with brain-specific deletions of Ink4a, Ink4b, Arf, or PTEN in logical combinations representing the progression of primary or secondary GBMs. Specifically, we examined whether complex, unrepairable DNA lesions, such as those induced by charged particles, are more tumorigenic compared to simple breaks that are efficiently repaired, such as those induced by gamma-rays. Using CDKN2A knockout murine astrocytes in which the tumor suppressors Ink4a and Arf have been deleted (abrogating RB1 and p53 pathways, respectively), we have previously shown that Fe ions are highly tumorigenic compared to gamma-rays. Importantly, we find that loss of a third tumor suppressor, Ink4b, is a critical event in tumorigenesis triggered by charged particles. Based on our in vitro results, we are now using transgenic mouse models with brain-specific deletions of these three key tumor suppressors (Ink4a/b, Arf) to evaluate radiation-induced gliomagenesis in vivo. Nestin-Cre; Ink4ab-/-; Arf f/f mice were irradiated with a single dose of 1 Gy Fe ions or 4 Gy gamma-rays. While gamma-induced damage is efficiently repaired by 1 day, unrepaired DNA lesions are evident up to 1 month following Fe irradiation. Most importantly, we find that combined loss of Ink4a/b and Arf cooperate with DNA damage by Fe ions resulting in a high incidence (25%) of malignant gliomas. These highly heterogeneous tumors arise with an average latency of 4.8 months and are classified as high grade (III and IV) glial tumors. Although tumors arising after gamma irradiation are also classified as high grade, they arise with a longer latency (5.6 months) and occur at a lower frequency (15%). To identify key genetic alterations involved in radiation-induced gliomagenesis, tumors are being analyzed by array CGH and microarray platforms. Strikingly, a 20- to 40-fold amplification of the MET proto-oncogene is observed in a high percentage of tumors. As MET is frequently amplified in human GBMs, it is likely that MET activation may be an important event in tumorigenesis triggered by charged particles in these mouse models. We are hopeful that a global and accurate picture of the genetic changes underlying radiation-induced gliomagenesis will emerge from this study. The data obtained will allow for comparison with changes recently identified in human gliomas by large scale genomic analyses and will allow us to understand whether radiation-induced gliomas are fundamentally similar to those occurring spontaneously, both in mouse models and in human patients.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2360. doi:1538-7445.AM2012-2360
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