Phytochemical Modulation of Ion Channels in Oncologic Symptomatology and Treatment.

Modern chemotherapies offer a broad approach to cancer treatment but eliminate both cancer and non-cancer cells indiscriminately and, thus, are associated with a host of side effects. Advances in precision oncology have brought about new targeted therapeutics, albeit mostly limited to a subset of patients with an actionable mutation. They too come with side effects and, ultimately, 'self-resistance' to the treatment. There is recent interest in the modulation of ion channels, transmembrane proteins that regulate the flow of electrically charged molecules in and out of cells, as an approach to aid treatment of cancer. Phytochemicals have been shown to act on ion channels with high specificity regardless of the tumor's genetic profile. This paper explores the use of phytochemicals in cancer symptom management and treatment.

GABA(A) receptor activation drives GABARAP-Nix mediated autophagy to radiation-sensitize primary and brain-metastatic lung adenocarcinoma tumors.

In non-small cell lung cancer (NSCLC) treatment, targeted therapies benefit only a subset of NSCLC, while radiotherapy responses are not durable and toxicity limits therapy. We find that a GABA(A) receptor activator, AM-101, impairs viability and clonogenicity of NSCLC primary and brain metastatic cells. Employing an ex vivo 'chip', AM-101 is as efficacious as the chemotherapeutic docetaxel, which is used with radiotherapy for advanced-stage NSCLC. In vivo , AM-101 potentiates radiation, including conferring a survival benefit to mice bearing NSCLC intracranial tumors. GABA(A) receptor activation stimulates a selective-autophagic response via multimerization of GABA(A) Receptor-Associated Protein (GABARAP), stabilization of mitochondrial receptor Nix, and utilization of ubiquitin-binding protein p62. A targeted-peptide disrupting Nix binding to GABARAP inhibits AM-101 cytotoxicity. This supports a model of GABA(A) receptor activation driving a GABARAP-Nix multimerization axis triggering autophagy. In patients receiving radiotherapy, GABA(A) receptor activation may improve tumor control while allowing radiation dose de-intensification to reduce toxicity. Highlights: Activating GABA(A) receptors intrinsic to lung primary and metastatic brain cancer cells triggers a cytotoxic response. GABA(A) receptor activation works as well as chemotherapeutic docetaxel in impairing lung cancer viability ex vivo . GABA(A) receptor activation increases survival of mice bearing lung metastatic brain tumors.A selective-autophagic response is stimulated by GABA(A) receptor activation that includes multimerization of GABARAP and Nix.Employing a new nanomolar affinity peptide that abrogates autophagosome formation inhibits cytotoxicity elicited by GABA(A) receptor activation.

Screening Ion Channels in Cancer Cells.

Ion channels are critical for cell development and maintaining cell homeostasis. The perturbation of ion channel function contributes to the development of a broad range of disorders or channelopathies. Cancer cells utilize ion channels to drive their own development, as well as to improve as a tumor and to assimilate in a microenvironment that includes various non-cancerous cells. Furthermore, increases in levels of growth factors and hormones within the tumor microenvironment can result in enhanced ion channel expression, which contributes to cancer cell proliferation and survival. Thus, the pharmacological targeting of ion channels is potentially a promising approach to treating solid malignancies, including primary and metastatic brain cancers. Herein, protocols to characterize the function of ion channels in cancerous cells and approaches to analyze modulators of ion channels to determine their impact on cancer viability are described. These include staining a cell(s) for an ion channel(s), testing the polarized state of mitochondria, establishing ion channel function using electrophysiology, and performing viability assays to assess drug potency.

Lessons learned from evolving frameworks in adult glioblastoma.

Glioblastoma (GBM) is the most common and aggressive malignant adult brain tumor. Significant effort has been directed to achieve a molecular subtyping of GBM to impact treatment. The discovery of new unique molecular alterations has resulted in a more effective classification of tumors and has opened the door to subtype-specific therapeutic targets. Morphologically identical GBM may have different genetic, epigenetic, and transcriptomic alterations and therefore different progression trajectories and response to treatments. With a transition to molecularly guided diagnosis, there is now a potential to personalize and successfully manage this tumor type to improve outcomes. The steps to achieve subtype-specific molecular signatures can be extrapolated to other neuroproliferative as well as neurodegenerative disorders.

Ligand-Gated Ion Channels as Targets for Treatment and Management of Cancers.

Ligand-gated ion channels are an ionotropic receptor subtype characterized by the binding of an extracellular ligand, followed by the transient passage of ions through a transmembrane pore. Ligand-gated ion channels are commonly subcategorized into three superfamilies: purinoreceptors, glutamate receptors, and Cys-loop receptors. This classification is based on the differing topographical morphology of the receptors, which in turn confers functional differences. Ligand-gated ion channels have a diverse spatial and temporal expression which implicate them in key cellular processes. Given that the transcellular electrochemical gradient is finely tuned in eukaryotic cells, any disruption in this homeostasis can contribute to aberrancies, including altering the activity of pro-tumorigenic molecular pathways, such as the MAPK/ERK, RAS, and mTOR pathways. Ligand-gated ion channels therefore serve as a potential targetable system for cancer therapeutics. In this review, we analyze the role that each of the three ligand-gated ion channel superfamilies has concerning tumor proliferation and as a target for the treatment of cancer symptomatology.

Reuse of Molecules for Glioblastoma Therapy.

Glioblastoma multiforme (GBM) is a highly malignant primary brain tumor. The current standard of care for GBM is the Stupp protocol which includes surgical resection, followed by radiotherapy concomitant with the DNA alkylator temozolomide; however, survival under this treatment regimen is an abysmal 12-18 months. New and emerging treatments include the application of a physical device, non-invasive 'tumor treating fields' (TTFs), including its concomitant use with standard of care; and varied vaccines and immunotherapeutics being trialed. Some of these approaches have extended life by a few months over standard of care, but in some cases are only available for a minority of GBM patients. Extensive activity is also underway to repurpose and reposition therapeutics for GBM, either alone or in combination with the standard of care. In this review, we present select molecules that target different pathways and are at various stages of clinical translation as case studies to illustrate the rationale for their repurposing-repositioning and potential clinical use.

Melanoma Cell Intrinsic GABAA Receptor Enhancement Potentiates Radiation and Immune Checkpoint Inhibitor Response by Promoting Direct and T Cell-Mediated Antitumor Activity.

PURPOSE: Most patients with metastatic melanoma show variable responses to radiation therapy and do not benefit from immune checkpoint inhibitors. Improved strategies for combination therapy that leverage potential benefits from radiation therapy and immune checkpoint inhibitors are critical. METHODS AND MATERIALS: We analyzed metastatic melanoma tumors in the TCGA cohort for expression of genes coding for subunits of type A γ-aminobutyric acid (GABA) receptor (GABAAR), a chloride ion channel and major inhibitory neurotransmitter receptor. Electrophysiology was used to determine whether melanoma cells possess intrinsic GABAAR activity. Melanoma cell viability studies were conducted to test whether enhancing GABAAR mediated chloride transport using benzodiazepine-impaired viability. A syngeneic melanoma mouse model was used to assay the effect of benzodiazepine on tumor volume and its ability to potentiate radiation therapy or immunotherapy. Treated tumors were analyzed for changes in gene expression by RNA sequencing and presence of tumor-infiltrating lymphocytes by flow cytometry. RESULTS: Genes coding for subunits of GABAARs express functional GABAARs in melanoma cells. By enhancing GABAAR-mediated anion transport, benzodiazepines depolarize melanoma cells and impair their viability. In vivo, benzodiazepine alone reduces tumor growth and potentiates radiation therapy and α-PD-L1 antitumor activity. The combination of benzodiazepine, radiation therapy, and α-PD-L1 results in near complete regression of treated tumors and a potent abscopal effect, mediated by increased infiltration of polyfunctional CD8+ T cells. Treated tumors show expression of cytokine-cytokine receptor interactions and overrepresentation of p53 signaling. CONCLUSIONS: This study identifies an antitumor strategy combining radiation and/or an immune checkpoint inhibitor with modulation of GABAARs in melanoma using benzodiazepine.

Epigenetics and survivorship in pediatric brain tumor patients.

INTRODUCTION: Brain tumors make up over a quarter of pediatric malignancies. Depending on the age of presentation and treatment, pediatric brain tumor survivors experience varying degrees of treatment induced morbidity and sequelae. Epigenetic mechanisms play a critical role in silencing of tumor suppressor genes and activation of driver genes involved in oncogenesis in different types of brain tumors. Epigenetic modifications in pediatric brain tumor patients may influence long-term survival and may refine the molecular response to treatment induced morbidity and sequelae. However, there is a dearth of studies on how epigenetics of pediatric brain tumors is connected with neurocognition and other treatment related sequelae in survivors. METHODS/RESULTS: In this review we explore epigenetic factors that may contribute to the survivorship and treatment of pediatric brain tumor patients. We focus on glioblastoma, medulloblastoma, and the neurocutaneous syndrome neurofibromatosis type-1 to highlight epigenetic biomarkers that can potentially serve not only as prognostic indicators of overall patient survival, but hopefully as indicators to the response to treatment neurocognitively and otherwise. CONCLUSIONS: Future studies will hopefully soon bridge the gap in our knowledge on how epigenetic modifications are linked to treatment related sequelae in pediatric brain tumor patients.

Impact of Sequencing Radiation Therapy and Immune Checkpoint Inhibitors in the Treatment of Melanoma Brain Metastases.

PURPOSE: Melanoma brain metastases (MBM) occur in ∼50% of melanoma patients. Although both radiation therapy (RT) and immune checkpoint inhibitor (ICI) are used alone or in combination for MBM treatment, the role of this combination and how these treatments could best be sequenced remains unclear. METHODS AND MATERIALS: We conducted a retrospective analysis of patients with resected MBM who underwent treatment with RT, ICI, or a combination of RT and ICI. Among the latter, we specifically investigated the differential gene expression via RNA-sequencing between patients who received RT first then ICI (RT → ICI) versus ICI first then RT (ICI → RT). We used a glycoprotein-transduced syngeneic melanoma mouse model for validation experiments. RESULTS: We found that for patients with resected MBM, a combination of RT and ICI confers superior survival compared with RT alone. Specifically, we found that RT → ICI was superior compared with ICI → RT. Transcriptome analysis of resected MBM revealed that the RT → ICI cohort demonstrated deregulation of genes involved in apoptotic signaling and key modulators of inflammation that are most implicated in nuclear factor kappa-light-chain-enhancer of activated B cells signaling. In a preclinical model, we showed that RT followed by anti-programmed death-ligand 1 therapy was superior to the reverse sequence of therapy, supporting the observations we made in patients with MBM. CONCLUSIONS: Our study provides initial insights into the optimal sequence of RT and ICI in the treatment of MBM after surgical resection. Prospective studies examining the best sequence of RT and ICI are necessary, and our study contributes to the rationale to pursue these.

Modulating native GABAA receptors in medulloblastoma with positive allosteric benzodiazepine-derivatives induces cell death.

PURPOSE: Pediatric brain cancer medulloblastoma (MB) standard-of-care results in numerous comorbidities. MB is comprised of distinct molecular subgroups. Group 3 molecular subgroup patients have the highest relapse rates and after standard-of-care have a 20% survival. Group 3 tumors have high expression of GABRA5, which codes for the α5 subunit of the γ-aminobutyric acid type A receptor (GABAAR). We are advancing a therapeutic approach for group 3 based on GABAAR modulation using benzodiazepine-derivatives. METHODS: We performed analysis of GABR and MYC expression in MB tumors and used molecular, cell biological, and whole-cell electrophysiology approaches to establish presence of a functional 'druggable' GABAAR in group 3 cells. RESULTS: Analysis of expression of 763 MB tumors reveals that group 3 tumors share high subgroup-specific and correlative expression of GABR genes, which code for GABAAR subunits α5, ß3 and γ2 and 3. There are ~ 1000 functional α5-GABAARs per group 3 patient-derived cell that mediate a basal chloride-anion efflux of 2 × 109 ions/s. Benzodiazepines, designed to prefer α5-GABAAR, impair group 3 cell viability by enhancing chloride-anion efflux with subtle changes in their structure having significant impact on potency. A potent, non-toxic benzodiazepine ('KRM-II-08') binds to the α5-GABAAR (0.8 µM EC50) enhancing a chloride-anion efflux that induces mitochondrial membrane depolarization and in response, TP53 upregulation and p53, constitutively phosphorylated at S392, cytoplasmic localization. This correlates with pro-apoptotic Bcl-2-associated death promoter protein localization. CONCLUSION: GABRA5 expression can serve as a diagnostic biomarker for group 3 tumors, while α5-GABAAR is a therapeutic target for benzodiazepine binding, enhancing an ion imbalance that induces apoptosis.

High-affinity gold nanoparticle pin to label and localize histidine-tagged protein in macromolecular assemblies.

There is significant demand for experimental approaches to aid protein localization in electron microscopy micrographs and ultimately in three-dimensional reconstructions of macromolecular assemblies. We report preparation and use of a reagent consisting of tris-nitrilotriacetic acid (tris-NTA) conjugated with a monofunctional gold nanoparticle ((AuNP)tris-NTA) for site-specific, non-covalent labeling of protein termini fused to a histidine-tag (His-tag). Multivalent binding of tris-NTA to a His-tag via complexed Ni(II) ions results in subnanomolar affinity and a defined 1:1 stoichiometry. Precise localization of (AuNP)tris-NTA labeled proteins by electron microscopy is further ensured by the reagent's short conformationally restricted linker. We used (AuNP)tris-NTA to localize His-tagged proteins in an oligomeric ATPase and in the bacterial 50S ribosomal subunit. (AuNP)tris-NTA can specifically bind to the target proteins in these assemblies and is clearly discernible. Our labeling reagent should find broad application in noncovalent, site-specific labeling of protein termini to pinpoint their location in macromolecular assemblies.

Medulloblastoma exome sequencing uncovers subtype-specific somatic mutations.

Medulloblastomas are the most common malignant brain tumours in children. Identifying and understanding the genetic events that drive these tumours is critical for the development of more effective diagnostic, prognostic and therapeutic strategies. Recently, our group and others described distinct molecular subtypes of medulloblastoma on the basis of transcriptional and copy number profiles. Here we use whole-exome hybrid capture and deep sequencing to identify somatic mutations across the coding regions of 92 primary medulloblastoma/normal pairs. Overall, medulloblastomas have low mutation rates consistent with other paediatric tumours, with a median of 0.35 non-silent mutations per megabase. We identified twelve genes mutated at statistically significant frequencies, including previously known mutated genes in medulloblastoma such as CTNNB1, PTCH1, MLL2, SMARCA4 and TP53. Recurrent somatic mutations were newly identified in an RNA helicase gene, DDX3X, often concurrent with CTNNB1 mutations, and in the nuclear co-repressor (N-CoR) complex genes GPS2, BCOR and LDB1. We show that mutant DDX3X potentiates transactivation of a TCF promoter and enhances cell viability in combination with mutant, but not wild-type, ß-catenin. Together, our study reveals the alteration of WNT, hedgehog, histone methyltransferase and now N-CoR pathways across medulloblastomas and within specific subtypes of this disease, and nominates the RNA helicase DDX3X as a component of pathogenic ß-catenin signalling in medulloblastoma.

Architecture of the spliceosome.

Precursor-mRNA splicing is catalyzed by an extraordinarily large and highly dynamic macromolecular assemblage termed the spliceosome. Detailed biochemical and structural study of the spliceosome presents a formidable challenge, but there has recently been significant progress made on this front highlighted by the crystal structure of a 10-subunit human U1 snRNP. This review provides an overview of our current understanding of the architecture of the spliceosome and the RNA-protein complexes integral to its function, the U snRNPs.

Crystal structure of human spliceosomal U1 snRNP at 5.5 A resolution.

Human spliceosomal U1 small nuclear ribonucleoprotein particles (snRNPs), which consist of U1 small nuclear RNA and ten proteins, recognize the 5' splice site within precursor messenger RNAs and initiate the assembly of the spliceosome for intron excision. An electron density map of the functional core of U1 snRNP at 5.5 A resolution has enabled us to build the RNA and, in conjunction with site-specific labelling of individual proteins, to place the seven Sm proteins, U1-C and U1-70K into the map. Here we present the detailed structure of a spliceosomal snRNP, revealing a hierarchical network of intricate interactions between subunits. A striking feature is the amino (N)-terminal polypeptide of U1-70K, which extends over a distance of 180 A from its RNA binding domain, wraps around the core domain consisting of the seven Sm proteins and finally contacts U1-C, which is crucial for 5'-splice-site recognition. The structure of U1 snRNP provides insights into U1 snRNP assembly and suggests a possible mechanism of 5'-splice-site recognition.