Malignant brain tumors are rapidly progressive and often fatal owing to resistance to therapies and based on their complex biology, heterogeneity, and isolation from systemic circulation. ...Glioblastoma is the most common and most aggressive primary brain tumor, has high mortality, and affects both children and adults. Despite significant advances in understanding the pathology, multiple clinical trials employing various treatment strategies have failed. With much expanded knowledge of the GBM genome, epigenome, and transcriptome, the field of neuro-oncology is getting closer to achieve breakthrough-targeted molecular therapies. Current developments of oligonucleotide chemistries for CNS applications make this new class of drugs very attractive for targeting molecular pathways dysregulated in brain tumors and are anticipated to vastly expand the spectrum of currently targetable molecules. In this chapter, we will overview the molecular landscape of malignant gliomas and explore the most prominent molecular targets (mRNAs, miRNAs, lncRNAs, and genomic mutations) that provide opportunities for the development of oligonucleotide therapeutics for this class of neurologic diseases. Because malignant brain tumors focally disrupt the blood–brain barrier, this class of diseases might be also more susceptible to systemic treatments with oligonucleotides than other neurologic disorders and, thus, present an entry point for the oligonucleotide therapeutics to the CNS. Nevertheless, delivery of oligonucleotides remains a crucial part of the treatment strategy. Finally, synthetic gRNAs guiding CRISPR–Cas9 editing technologies have a tremendous potential to further expand the applications of oligonucleotide therapeutics and take them beyond RNA targeting.
Venous thromboembolism occurs in up to one-third of patients with primary brain tumors. Spontaneous intracranial hemorrhage (ICH) is also a frequent occurrence in these patients, but there is limited ...data on the safety of therapeutic anticoagulation. To determine the rate of ICH in patients treated with enoxaparin, we performed a matched, retrospective cohort study with blinded radiology review for 133 patients with high-grade glioma. After diagnosis of glioma, the cohort that received enoxaparin was 3 times more likely to develop a major ICH than those not treated with anticoagulation (14.7% vs 2.5%; P = .036; hazard ratio HR, 3.37; 95% confidence interval CI, 1.02-11.14). When enoxaparin was analyzed as a time-varying covariate, anticoagulation was associated with a >13-fold increased risk of hemorrhage (HR, 13.26; 95% CI, 3.33-52.85; P < .0001). Overall survival was significantly shorter for patients who suffered a major ICH on enoxaparin compared with patients not receiving anticoagulation (3.3 vs 10.2 months; log-rank P = .012). We applied a validated ICH prediction risk score PANWARDS (platelets, albumin, no congestive heart failure, warfarin, age, race, diastolic blood pressure, stroke), and observed that all major ICHs on enoxaparin occurred in the setting of a PANWARDS score ≥25, corresponding with a sensitivity of 100% (95% CI, 63% to 100%) and a specificity of 40% (95% CI, 25% to 56%). We conclude that caution is warranted when considering therapeutic anticoagulation in patients with high-grade gliomas given the increased risk of ICH and poor prognosis after a major hemorrhage on anticoagulation. The PANWARDS score may assist clinicians in identifying the patients at greatest risk of suffering a major intracranial hemorrhage with anticoagulation.
•Therapeutic enoxaparin was associated with a greater than threefold increased risk of major ICH in patients with glioma.•The PANWARDS risk score was a sensitive predictor of major ICH in glioma.
Glioblastoma (GBM) brain tumor remains among the most lethal and incurable human diseases. Oncogenic microRNA-10b (miR-10b) is strongly and universally upregulated in GBM, and its inhibition by ...antisense oligonucleotides (ASOs) reduces the growth of heterogeneous glioma cells; therefore, miR-10b represents a unique therapeutic target for GBM. Here we explored the effects of miR-10b gene editing on GBM. Using the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system, we investigated effects of miR-10b gene editing on the growth of cultured human glioma cells, tumor-initiating stem-like cells, and mouse GBM xenografts, as well as the oncogene-induced transformation of normal astrocytes. We show that GBM is strictly “addicted” to miR-10b and that miR-10b gene ablation is lethal for glioma cell cultures and established intracranial tumors. miR-10b loss-of-function mutations lead to the death of glioma, but not other cancer cell lines. We have not detected escaped proliferative clones of GBM cells edited in the miR-10b locus. Finally, neoplastic transformation of normal astrocytes was abolished by the miR-10b-editing vectors. This study demonstrates the feasibility of gene editing for brain tumors in vivo and suggests virus-mediated miR-10b gene ablation as a promising therapeutic approach that permanently eliminates the key regulator essential for tumor growth and survival.
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Oncogenic microRNA-10b (miR-10b) is strongly and universally upregulated in glioblastoma brain tumors. In this issue of Molecular Therapy, El Fatimy and colleagues show that glioblastoma is addicted to miR-10b and that miR-10b gene editing by viral vectors presents a new therapeutic approach for malignant gliomas.
Highlights • Brain metastases are frequent in ALK rearranged lung cancers. • Brain metastases are frequent in EGFR mutated lung cancers. • ∼25% of patients at diagnosis and half at 3-years of ...survival have brain metastases.
Glioblastoma (GBM) is an incurable primary malignant brain cancer hallmarked with a substantial protumorigenic immune component. Knowledge of the GBM immune microenvironment during tumor evolution ...and standard of care treatments is limited. Using single-cell transcriptomics and flow cytometry, we unveiled large-scale comprehensive longitudinal changes in immune cell composition throughout tumor progression in an epidermal growth factor receptor-driven genetic mouse GBM model. We identified subsets of proinflammatory microglia in developing GBMs and anti-inflammatory macrophages and protumorigenic myeloid-derived suppressors cells in end-stage tumors, an evolution that parallels breakdown of the blood-brain barrier and extensive growth of epidermal growth factor receptor
GBM cells. A similar relationship was found between microglia and macrophages in patient biopsies of low-grade glioma and GBM. Temozolomide decreased the accumulation of myeloid-derived suppressor cells, whereas concomitant temozolomide irradiation increased intratumoral GranzymeB
CD8
T cells but also increased CD4
regulatory T cells. These results provide a comprehensive and unbiased immune cellular landscape and its evolutionary changes during GBM progression.
Venous thromboembolism occurs frequently in patients with cancer who have brain metastases, but there is limited evidence supporting the safety of therapeutic anticoagulation. To assess the risk for ...intracranial hemorrhage associated with the administration of therapeutic doses of low-molecular-weight heparin, we performed a matched, retrospective cohort study of 293 patients with cancer with brain metastases (104 with therapeutic enoxaparin and 189 controls). A blinded review of radiographic imaging was performed, and intracranial hemorrhages were categorized as trace, measurable, and significant. There were no differences observed in the cumulative incidence of intracranial hemorrhage at 1 year in the enoxaparin and control cohorts for measurable (19% vs 21%; Gray test, P = .97; hazard ratio, 1.02; 90% confidence interval CI, 0.66-1.59), significant (21% vs 22%; P = .87), and total (44% vs 37%; P = .13) intracranial hemorrhages. The risk for intracranial hemorrhage was fourfold higher (adjusted hazard ratio, 3.98; 90% CI, 2.41-6.57; P < .001) in patients with melanoma or renal cell carcinoma (N = 60) than lung cancer (N = 153), but the risk was not influenced by the administration of enoxaparin. Overall survival was similar for the enoxaparin and control cohorts (8.4 vs 9.7 months; Log-rank, P = .65). We conclude that intracranial hemorrhage is frequently observed in patients with brain metastases, but that therapeutic anticoagulation does not increase the risk for intracranial hemorrhage.
•Significant intracranial hemorrhage occurs in 20% to 50% of patients with metastatic brain tumors.•Therapeutic anticoagulation in patients with brain metastasis did not increase the risk for intracranial hemorrhage.
MicroRNA‐10b (miR‐10b) is a unique oncogenic miRNA that is highly expressed in all GBM subtypes, while absent in normal neuroglial cells of the brain. miR‐10b inhibition strongly impairs ...proliferation and survival of cultured glioma cells, including glioma‐initiating stem‐like cells (GSC). Although several miR‐10b targets have been identified previously, the common mechanism conferring the miR‐10b‐sustained viability of GSC is unknown. Here, we demonstrate that in heterogeneous GSC, miR‐10b regulates cell cycle and alternative splicing, often through the non‐canonical targeting via 5′UTRs of its target genes, including MBNL1‐3, SART3, and RSRC1. We have further assessed the inhibition of miR‐10b in intracranial human GSC‐derived xenograft and murine GL261 allograft models in athymic and immunocompetent mice. Three delivery routes for the miR‐10b antisense oligonucleotide inhibitors (ASO), direct intratumoral injections, continuous osmotic delivery, and systemic intravenous injections, have been explored. In all cases, the treatment with miR‐10b ASO led to targets’ derepression, and attenuated growth and progression of established intracranial GBM. No significant systemic toxicity was observed upon ASO administration by local or systemic routes. Our results indicate that miR‐10b is a promising candidate for the development of targeted therapies against all GBM subtypes.
Synopsis
Oncogenic miR‐10b highly expressed in glioblastoma and absent in normal brain tissues promotes the growth of heterogeneous glioma cell types. Teplyuk et al. describe common genes regulated by miR‐10b in glioma stem cells and demonstrate the efficacy of miR‐10b inhibition in established GBM models.
mRNA splicing and processing factors are regulated by miR‐10b through the non‐canonical 5′UTR binding in glioma‐derived neurospheres.
Growth of orthotopic glioblastoma (GBM) xenografts and allografts in mouse models is attenuated by local and systemic delivery of miR+NA inhibitors.
No systemic toxicity of miR‐10b inhibitors’ administration by local or systemic routes was observed.
Oncogenic miR‐10b highly expressed in glioblastoma and absent in normal brain tissues promotes the growth of heterogeneous glioma cell types. Teplyuk et al. describe common genes regulated by miR‐10b in glioma stem cells and demonstrate the efficacy of miR‐10b inhibition in established GBM models.
Abstract
Meningioma is the most common primary central nervous system tumor. Although mostly nonmalignant, meningioma can cause serious complications by mass effect and vasogenic edema. While surgery ...and radiation improve outcomes, not all cases can be treated due to eloquent location. Presently no medical treatment is available to slow meningioma growth owing to incomplete understanding of the underlying pathology, which in turn is due to the lack of high-fidelity tissue culture and animal models. We propose a simple and rapid method for the establishment of meningioma tumor-derived primary cultures. These cells can be maintained in culture for a limited time in serum-free media as spheres and form adherent cultures in the presence of 4% fetal calf serum. Many of the tissue samples show expression of the lineage marker PDG2S, which is typically retained in matched cultured cells, suggesting the presence of cells of arachnoid origin. Furthermore, nonarachnoid cells including vascular endothelial cells are also present in the cultures in addition to arachnoid cells, potentially providing a more accurate tumor cell microenvironment, and thus making the model more relevant for meningioma research and high-throughput drug screening.
miRNAs are regulatory transcripts established as repressors of mRNA stability and translation that have been functionally implicated in carcinogenesis. miR-10b is one of the key onco-miRs associated ...with multiple forms of cancer. Malignant gliomas exhibit particularly striking dependence on miR-10b. However, despite the therapeutic potential of miR-10b targeting, this miRNA's poorly investigated and largely unconventional properties hamper the clinical translation.
We utilized Covalent Ligation of Endogenous Argonaute-bound RNAs and their high-throughput RNA sequencing to identify miR-10b interactome and a combination of biochemical and imaging approaches for target validation. They included Crosslinking and RNA immunoprecipitation with spliceosomal proteins, a combination of miRNA FISH with protein immunofluorescence in glioma cells and patient-derived tumors, native Northern blotting, and the transcriptome-wide analysis of alternative splicing.
We demonstrate that miR-10b binds to U6 snRNA, a core component of the spliceosomal machinery. We provide evidence of the direct binding between miR-10b and U6, in situ imaging of miR-10b and U6 co-localization in glioma cells and tumors, and biochemical co-isolation of miR-10b with the components of the spliceosome. We further demonstrate that miR-10b modulates U6 N-6-adenosine methylation and pseudouridylation, U6 binding to splicing factors SART3 and PRPF8, and regulates U6 stability, conformation, and levels. These effects on U6 result in global splicing alterations, exemplified by the altered ratio of the isoforms of a small GTPase CDC42, reduced overall CDC42 levels, and downstream CDC42 -mediated effects on cell viability.
We identified U6 snRNA, the key RNA component of the spliceosome, as the top miR-10b target in glioblastoma. We, therefore, present an unexpected intersection of the miRNA and splicing machineries and a new nuclear function for a major cancer-associated miRNA.
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