Preclinical systemic pharmacokinetics, dose-proportionality, and CNS distribution of the ATM inhibitor WSD0628, a novel radiosensitizer for the treatment of brain tumors.

Radiation therapy, a standard treatment option for many cancer patients, induces DNA double strand breaks (DSBs), leading to cell death. Ataxia telangiectasia mutated (ATM) kinase is a key regulator of DSB repair, and ATM inhibitors are being explored as radiosensitizers for various tumors, including primary and metastatic brain tumors. Efficacy of radiosensitizers for brain tumors may be influenced by a lack of effective drug delivery across the blood-brain barrier (BBB). The objective of this study was to evaluate the systemic pharmacokinetics and mechanisms that influence the CNS distribution of WSD0628, a novel and potent ATM inhibitor, in the mouse. Further, we have used these observations to form the basis of predicting effective exposures for clinical application. We observed a greater than dose proportional increase in exposure, likely due to saturation of clearance processes. Our results show that WSD0628 is orally bioavailable and CNS penetrant, with unbound partitioning in CNS (i.e., Kpuu) between 0.15 and 0.3. CNS distribution is not limited by the efflux transporters P-gp and Bcrp. WSD0628 is distributed uniformly amongst different brain regions. Thus, WSD0628 has favorable pharmacokinetic properties and potential for further exploration to determine the PK-PD-efficacy relationship in CNS tumors. This approach will provide critical insights for the clinical translation of WSD0628 for the treatment of primary and secondary brain tumors. Significance Statement This study evaluates the preclinical systemic pharmacokinetics, dose proportionality, and mechanisms influencing CNS distribution of WSD0628, a novel ATM inhibitor for the treatment of brain tumors. Results indicate that WSD0628 is orally bioavailable and CNS penetrant without efflux transporter liability. We also observed a greater than dose-proportional increase in exposure in both the plasma and brain. These favorable pharmacokinetic properties indicate WSD0628 has potential for further exploration for use as a radiosensitizer in the treatment of brain tumors.

massNet: integrated processing and classification of spatially resolved mass spectrometry data using deep learning for rapid tumor delineation.

MOTIVATION: Mass spectrometry imaging (MSI) provides rich biochemical information in a label-free manner and therefore holds promise to substantially impact current practice in disease diagnosis. However, the complex nature of MSI data poses computational challenges in its analysis. The complexity of the data arises from its large size, high-dimensionality and spectral nonlinearity. Preprocessing, including peak picking, has been used to reduce raw data complexity; however, peak picking is sensitive to parameter selection that, perhaps prematurely, shapes the downstream analysis for tissue classification and ensuing biological interpretation. RESULTS: We propose a deep learning model, massNet, that provides the desired qualities of scalability, nonlinearity and speed in MSI data analysis. This deep learning model was used, without prior preprocessing and peak picking, to classify MSI data from a mouse brain harboring a patient-derived tumor. The massNet architecture established automatically learning of predictive features, and automated methods were incorporated to identify peaks with potential for tumor delineation. The model's performance was assessed using cross-validation, and the results demonstrate higher accuracy and a substantial gain in speed compared to the established classical machine learning method, support vector machine. AVAILABILITY AND IMPLEMENTATION: https://github.com/wabdelmoula/massNet. The data underlying this article are available in the NIH Common Fund's National Metabolomics Data Repository (NMDR) Metabolomics Workbench under project id (PR001292) with http://dx.doi.org/10.21228/M8Q70T. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Salicylates enhance CRM1 inhibitor antitumor activity by induction of S-phase arrest and impairment of DNA-damage repair.

Chromosome region maintenance protein 1 (CRM1) mediates protein export from the nucleus and is a new target for anticancer therapeutics. Broader application of KPT-330 (selinexor), a first-in-class CRM1 inhibitor recently approved for relapsed multiple myeloma and diffuse large B-cell lymphoma, have been limited by substantial toxicity. We discovered that salicylates markedly enhance the antitumor activity of CRM1 inhibitors by extending the mechanisms of action beyond CRM1 inhibition. Using salicylates in combination enables targeting of a range of blood cancers with a much lower dose of selinexor, thereby potentially mitigating prohibitive clinical adverse effects. Choline salicylate (CS) with low-dose KPT-330 (K+CS) had potent, broad activity across high-risk hematological malignancies and solid-organ cancers ex vivo and in vivo. The K+CS combination was not toxic to nonmalignant cells as compared with malignant cells and was safe without inducing toxicity to normal organs in mice. Mechanistically, compared with KPT-330 alone, K+CS suppresses the expression of CRM1, Rad51, and thymidylate synthase proteins, leading to more efficient inhibition of CRM1-mediated nuclear export, impairment of DNA-damage repair, reduced pyrimidine synthesis, cell-cycle arrest in S-phase, and cell apoptosis. Moreover, the addition of poly (ADP-ribose) polymerase inhibitors further potentiates the K+CS antitumor effect. K+CS represents a new class of therapy for multiple types of blood cancers and will stimulate future investigations to exploit DNA-damage repair and nucleocytoplasmic transport for cancer therapy in general.

How Much is Enough? Impact of Efflux Transporters on Drug delivery Leading to Efficacy in the Treatment of Brain Tumors.

The lack of effective chemotherapeutic agents for the treatment of brain tumors is a serious unmet medical need. This can be attributed, in part, to inadequate delivery through the blood-brain barrier (BBB) and the tumor-cell barrier, both of which have active efflux transporters that can restrict the transport of many potentially effective agents for both primary and metastatic brain tumors. This review briefly summarizes the components and function of the normal BBB with respect to drug penetration into the brain and the alterations in the BBB due to brain tumor that could influence drug delivery. Depending on what is rate-limiting a compound's distribution, the limited permeability across the BBB and the subsequent delivery into the tumor cell can be greatly influenced by efflux transporters and these are discussed in some detail. Given these complexities, it is necessary to quantify the extent of brain distribution of the active (unbound) drug to compare across compounds and to inform potential for use against brain tumors. In this regard, the metric, Kp,uu, a brain-to-plasma unbound partition coefficient, is examined and its current use is discussed. However, the extent of active drug delivery is not the only determinant of effective therapy. In addition to Kp,uu, drug potency is an important parameter that should be considered alongside drug delivery in drug discovery and development processes. In other words, to answer the question - How much is enough? - one must consider how much can be delivered with how much needs to be delivered.

Differential H2O2 Metabolism among Glioblastoma Subtypes Confers Variable Responses to Pharmacological Ascorbate Therapy Combined with Chemoradiation.

Glioblastoma (GBM), a highly lethal and aggressive central nervous system malignancy, presents a critical need for targeted therapeutic approaches to improve patient outcomes in conjunction with standard-of-care (SOC) treatment. Molecular subtyping based on genetic profiles and metabolic characteristics has advanced our understanding of GBM to better predict its evolution, mechanisms, and treatment regimens. Pharmacological ascorbate (P-AscH-) has emerged as a promising supplementary cancer therapy, leveraging its pro-oxidant properties to selectively kill malignant cells when combined with SOC. Given the clinical challenges posed by the heterogeneity and resistance of various GBM subtypes to conventional SOC, our study assessed the response of classical, mesenchymal, and proneural GBM to P-AscH-. P-AscH- (20 pmol/cell) combined with SOC (5 µM temozolomide and 4 Gy of radiation) enhanced clonogenic cell killing in classical and mesenchymal GBM subtypes, with limited effects in the proneural subtype. Similarly, following exposure to P-AscH- (20 pmol/cell), single-strand DNA damage significantly increased in classical and mesenchymal but not proneural GBM. Moreover, proneural GBM exhibited increased hydrogen peroxide removal rates, along with increased catalase and glutathione peroxidase activities compared to mesenchymal and classical GBM, demonstrating an altered H2O2 metabolism that potentially drives differential P-AscH- toxicity. Taken together, these data suggest that P-AscH- may hold promise as an approach to improve SOC responsiveness in mesenchymal GBMs that are known for their resistance to SOC.

The influence of the blood-brain barrier in the treatment of brain tumours.

Brain tumours have a poor prognosis and lack effective treatments. The blood-brain barrier (BBB) represents a major hurdle to drug delivery to brain tumours. In some locations in the tumour, the BBB may be disrupted to form the blood-brain tumour barrier (BBTB). This leaky BBTB enables diagnosis of brain tumours by contrast enhanced magnetic resonance imaging; however, this disruption is heterogeneous throughout the tumour. Thus, relying on the disrupted BBTB for achieving effective drug concentrations in brain tumours has met with little clinical success. Because of this, it would be beneficial to design drugs and drug delivery strategies to overcome the 'normal' BBB to effectively treat the brain tumours. In this review, we discuss the role of BBB/BBTB in brain tumour diagnosis and treatment highlighting the heterogeneity of the BBTB. We also discuss various strategies to improve drug delivery across the BBB/BBTB to treat both primary and metastatic brain tumours. Recognizing that the BBB represents a critical determinant of drug efficacy in central nervous system tumours will allow a more rapid translation from basic science to clinical application. A more complete understanding of the factors, such as BBB-limited drug delivery, that have hindered progress in treating both primary and metastatic brain tumours, is necessary to develop more effective therapies.

Central Nervous System Delivery of the Catalytic Subunit of DNA-Dependent Protein Kinase Inhibitor Peposertib as Radiosensitizer for Brain Metastases.

Cytotoxic effects of chemotherapy and radiation therapy (RT) used for the treatment of brain metastases results from DNA damage within cancer cells. Cells rely on highly evolved DNA damage response (DDR) pathways to repair the damage caused by these treatments. Inhibiting these repair pathways can further sensitize cancer cells to chemotherapy and RT. The catalytic subunit of DNA-dependent protein kinase, in a complex with Ku80 and Ku70, is a pivotal regulator of the DDR, and peposertib is a potent inhibitor of this catalytic subunit. The characterization of central nervous system (CNS) distributional kinetics of peposertib is critical in establishing a therapeutic index in the setting of brain metastases. Our studies demonstrate that the delivery of peposertib is severely restricted into the CNS as opposed to peripheral organs, by active efflux at the blood-brain barrier (BBB). Peposertib has a low free fraction in the brain and spinal cord, further reducing the active concentration, and distributes to the same degree within different anatomic regions of the brain. However, peposertib is heterogeneously distributed within the metastatic tumor, where its concentration is highest within the tumor core (with disrupted BBB) and substantially lower within the invasive tumor rim (with a relatively intact BBB) and surrounding normal brain. These findings are critical in guiding the potential clinical deployment of peposertib as a radiosensitizing agent for the safe and effective treatment of brain metastases. SIGNIFICANCE STATEMENT: Effective radiosensitization of brain metastases while avoiding toxicity to the surrounding brain is critical in the development of novel radiosensitizers. The central nervous system distribution of peposertib, a potent catalytic subunit of DNA-dependent protein kinase inhibitor, is restricted by active efflux in the normal blood-brain barrier (BBB) but can reach significant concentrations in the tumor core. This finding suggests that peposertib may be an effective radiosensitizer for intracranial tumors with an open BBB, while limited distribution into normal brain will decrease the risk of enhanced radiation injury.

Central Nervous System Distribution of the Ataxia-Telangiectasia Mutated Kinase Inhibitor AZD1390: Implications for the Treatment of Brain Tumors.

Effective drug delivery to the brain is critical for the treatment of glioblastoma (GBM), an aggressive and invasive primary brain tumor that has a dismal prognosis. Radiation therapy, the mainstay of brain tumor treatment, works by inducing DNA damage. Therefore, inhibiting DNA damage response (DDR) pathways can sensitize tumor cells to radiation and enhance cytotoxicity. AZD1390 is an inhibitor of ataxia-telangiectasia mutated kinase, a critical regulator of DDR. Our in vivo studies in the mouse indicate that delivery of AZD1390 to the central nervous system (CNS) is restricted due to active efflux by P-glycoprotein (P-gp). The free fraction of AZD1390 in brain and spinal cord were found to be low, thereby reducing the partitioning of free drug to these organs. Coadministration of an efflux inhibitor significantly increased CNS exposure of AZD1390. No differences were observed in distribution of AZD1390 within different anatomic regions of CNS, and the functional activity of P-gp and breast cancer resistance protein also remained the same across brain regions. In an intracranial GBM patient-derived xenograft model, AZD1390 accumulation was higher in the tumor core and rim compared with surrounding brain. Despite this heterogenous delivery within tumor-bearing brain, AZD1390 concentrations in normal brain, tumor rim, and tumor core were above in vitro effective radiosensitizing concentrations. These results indicate that despite being a substrate of efflux in the mouse brain, sufficient AZD1390 exposure is anticipated even in regions of normal brain. SIGNIFICANCE STATEMENT: Given the invasive nature of glioblastoma (GBM), tumor cells are often protected by an intact blood-brain barrier, requiring the development of brain-penetrant molecules for effective treatment. We show that efflux mediated by P-glycoprotein (P-gp) limits central nervous system (CNS) distribution of AZD1390 and that there are no distributional differences within anatomical regions of CNS. Despite efflux by P-gp, concentrations effective for potent radiosensitization are achieved in GBM tumor-bearing mouse brains, indicating that AZD1390 is an attractive molecule for clinical development of brain tumors.

Factors Influencing Luciferase-Based Bioluminescent Imaging in Preclinical Models of Brain Tumor.

Bioluminescent imaging (BLI) is a powerful tool in biomedical research to measure gene expression and tumor growth. The current study examined factors that influence the BLI signal, specifically focusing on the tissue distribution of two luciferase substrates, D-luciferin and CycLuc1. D-luciferin, a natural substrate of firefly luciferase, has been reported to have limited brain distribution, possibly due to the efflux transporter, breast cancer resistance protein (Bcrp), at the blood-brain barrier. CycLuc1, a synthetic analog of D-luciferin, has a greater BLI signal at lower doses than D-luciferin, especially in the brain. Our results indicate that limited brain distribution of D-luciferin and CycLuc1 is predominantly dictated by their low intrinsic permeability across the cell membrane, where the efflux transporter, Bcrp, plays a relatively minor role. Both genetic ablation and pharmacological inhibition of Bcrp decreased the systemic clearance of both luciferase substrates, significantly increasing exposure in the blood and, hence, in organs and tissues. These data also indicate that the biodistribution of luciferase substrates can be differentially influenced in luciferase-bearing tissues, leading to a "tissue-dependent" BLI signal. The results of this study point to the need to consider multiple mechanisms that influence the distribution of luciferase substrates. SIGNIFICANCE STATEMENT: Bioluminescence is used to monitor many biological processes, including tumor growth. This study examined the pharmacokinetics, brain distribution, and the role of active efflux transporters on the luciferase substrates D-luciferin and CycLuc1. CycLuc1 has a more sustained systemic circulation time (longer half-life) that can provide an advantage for the superior imaging outcome of CycLuc1 over D-luciferin. The disparity in imaging intensities between brain and peripheral sites is due to low intrinsic permeability of these luciferase substrates across the blood-brain barrier.

Nanocell-mediated delivery of miR-34a counteracts temozolomide resistance in glioblastoma.

BACKGROUND: Glioblastoma is the most common primary brain tumor and remains uniformly fatal, highlighting the dire need for developing effective therapeutics. Significant intra- and inter-tumor heterogeneity and inadequate delivery of therapeutics across blood-brain barrier continue to be significant impediments towards developing therapies which can significantly enhance survival. We hypothesize that microRNAs have the potential to serve as effective therapeutics for glioblastoma as they modulate the activity of multiple signaling pathways, and hence can counteract heterogeneity if successfully delivered. METHODS: Using a computational approach, we identified microRNA-34a as a microRNA that maximally reduces the activation status of the three core signaling networks (the receptor tyrosine kinase, p53 and Rb networks) that have been found to be deregulated in most glioblastoma tumors. Glioblastoma cultures were transfected with microRNA-34a or control microRNA to assess biological function and therapeutic potential in vitro. Nanocells were derived from genetically modified bacteria and loaded with microRNA-34a for intravenous administration to orthotopic patient-derived glioblastoma xenografts in mice. RESULTS: Overexpression of microRNA-34a strongly reduced the activation status of the three core signaling networks. microRNA-34a transfection also inhibited the survival of multiple established glioblastoma cell lines, as well as primary patient-derived xenograft cultures representing the proneural, mesenchymal and classical subtypes. Transfection of microRNA-34a enhanced temozolomide (TMZ) response in in vitro cultures of glioblastoma cells with primary TMZ sensitivity, primary TMZ resistance and acquired TMZ resistance. Mechanistically, microRNA-34a downregulated multiple therapeutic resistance genes which are associated with worse survival in glioblastoma patients and are enriched in specific tumor spatial compartments. Importantly, intravenous administration of nanocells carrying miR-34a and targeted to epidermal growth factor receptor (EGFR) strongly enhanced TMZ sensitivity in an orthotopic patient-derived xenograft mouse model of glioblastoma. CONCLUSIONS: Targeted bacterially-derived nanocells are an effective vehicle for the delivery of microRNA-34a to glioblastoma tumors. microRNA-34a inhibits survival and strongly sensitizes a wide range of glioblastoma cell cultures to TMZ, suggesting that combination therapy of TMZ with microRNA-34a loaded nanocells may serve as a novel therapeutic approach for the treatment of glioblastoma tumors.

Brain Distribution of Berzosertib: An Ataxia Telangiectasia and Rad3-Related Protein Inhibitor for the Treatment of Glioblastoma.

The effective treatment of brain tumors is a considerable challenge in part because of the presence of the blood-brain barrier (BBB) that limits drug delivery. Glioblastoma multiforme (GBM) is an aggressive and infiltrative primary brain tumor with an extremely poor prognosis after standard-of-care therapy with surgery, radiotherapy (RT), and chemotherapy. DNA damage response (DDR) pathways play a critical role in DNA repair in cancer cells, and inhibition of these pathways can potentially augment RT and chemotherapy tumor cell toxicity. The ataxia telangiectasia and Rad3-related protein (ATR) kinase is a key regulator of the DDR network and is potently and selectively inhibited by the ATR inhibitor berzosertib. Although in vitro studies demonstrate a synergistic effect of berzosertib in combination with temozolomide, in vivo efficacy studies have yet to recapitulate this observation using intracranial tumor models. In the current study, we demonstrate that delivery of berzosertib to the brain is restricted by efflux at the BBB. Berzosertib has a high binding affinity to brain tissue compared with plasma, thereby leading to low free drug concentrations in the brain. Berzosertib distribution is heterogenous within the tumor, wherein concentrations are substantially lower in normal brain and invasive tumor rim (wherein the BBB is intact) when compared with those in the tumor core (wherein the BBB is leaky). These results demonstrate that high tissue binding and limited and heterogenous brain distribution of berzosertib may be important factors that influence the efficacy of berzosertib therapy in GBM. SIGNIFICANCE STATEMENT: This study examined the brain delivery and efficacy of berzosertib in patient-derived xenograft models of glioblastoma multiforme (GBM). Berzosertib is actively effluxed at the blood-brain barrier and is highly bound to brain tissue, leading to low free drug concentrations in the brain. Berzosertib is heterogeneously distributed into different regions of the brain and tumor and, in this study, was not efficacious in vivo when combined with temozolomide. These factors inform the future clinical utility of berzosertib for GBM.

Synthetic and systems biology principles in the design of programmable oncolytic virus immunotherapies for glioblastoma.

Oncolytic viruses (OVs) are a class of immunotherapeutic agents with promising preclinical results for the treatment of glioblastoma (GBM) but have shown limited success in recent clinical trials. Advanced bioengineering principles from disciplines such as synthetic and systems biology are needed to overcome the current challenges faced in developing effective OV-based immunotherapies for GBMs, including off-target effects and poor clinical responses. Synthetic biology is an emerging field that focuses on the development of synthetic DNA constructs that encode networks of genes and proteins (synthetic genetic circuits) to perform novel functions, whereas systems biology is an analytical framework that enables the study of complex interactions between host pathways and these synthetic genetic circuits. In this review, the authors summarize synthetic and systems biology concepts for developing programmable, logic-based OVs to treat GBMs. Programmable OVs can increase selectivity for tumor cells and enhance the local immunological response using synthetic genetic circuits. The authors discuss key principles for developing programmable OV-based immunotherapies, including how to 1) select an appropriate chassis, a vector that carries a synthetic genetic circuit, and 2) design a synthetic genetic circuit that can be programmed to sense key signals in the GBM microenvironment and trigger release of a therapeutic payload. To illustrate these principles, some original laboratory data are included, highlighting the need for systems biology studies, as well as some preliminary network analyses in preparation for synthetic biology applications. Examples from the literature of state-of-the-art synthetic genetic circuits that can be packaged into leading candidate OV chassis are also surveyed and discussed.

Crosstalk between microglia and patient-derived glioblastoma cells inhibit invasion in a three-dimensional gelatin hydrogel model.

BACKGROUND: Glioblastoma is the most common and deadly form of primary brain cancer, accounting for more than 13,000 new diagnoses annually in the USA alone. Microglia are the innate immune cells within the central nervous system, acting as a front-line defense against injuries and inflammation via a process that involves transformation from a quiescent to an activated phenotype. Crosstalk between GBM cells and microglia represents an important axis to consider in the development of tissue engineering platforms to examine pathophysiological processes underlying GBM progression and therapy. METHODS: This work used a brain-mimetic hydrogel system to study patient-derived glioblastoma specimens and their interactions with microglia. Here, glioblastoma cells were either cultured alone in 3D hydrogels or in co-culture with microglia in a manner that allowed secretome-based signaling but prevented direct GBM-microglia contact. Patterns of GBM cell invasion were quantified using a three-dimensional spheroid assay. Secretome and transcriptome (via RNAseq) were used to profile the consequences of GBM-microglia interactions. RESULTS: Microglia displayed an activated phenotype as a result of GBM crosstalk. Three-dimensional migration patterns of patient-derived glioblastoma cells showed invasion was significantly decreased in response to microglia paracrine signaling. Potential molecular mechanisms underlying with this phenotype were identified from bioinformatic analysis of secretome and RNAseq data. CONCLUSION: The data demonstrate a tissue engineered hydrogel platform can be used to investigate crosstalk between immune cells of the tumor microenvironment related to GBM progression. Such multi-dimensional models may provide valuable insight to inform therapeutic innovations to improve GBM treatment.

Semaphorin 3A mediated brain tumor stem cell proliferation and invasion in EGFRviii mutant gliomas.

BACKGROUND: Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, with a median survival of approximately 15 months. Semaphorin 3A (Sema3A), known for its axon guidance and antiangiogenic properties, has been implicated in GBM growth. We hypothesized that Sema3A directly inhibits brain tumor stem cell (BTSC) proliferation and drives invasion via Neuropilin 1 (Nrp1) and Plexin A1 (PlxnA1) receptors. METHODS: GBM BTSC cell lines were assayed by immunostaining and PCR for levels of Semaphorin 3A (Sema3A) and its receptors Nrp1 and PlxnA1. Quantitative BrdU, cell cycle and propidium iodide labeling assays were performed following exogenous Sema3A treatment. Quantitative functional 2-D and 3-D invasion assays along with shRNA lentiviral knockdown of Nrp1 and PlxnA1 are also shown. In vivo flank studies comparing tumor growth of knockdown versus control BTSCs were performed. Statistics were performed using GraphPad Prism v7. RESULTS: Immunostaining and PCR analysis revealed that BTSCs highly express Sema3A and its receptors Nrp1 and PlxnA1, with expression of Nrp1 in the CD133 positive BTSCs, and absence in differentiated tumor cells. Treatment with exogenous Sema3A in quantitative BrdU, cell cycle, and propidium iodide labeling assays demonstrated that Sema3A significantly inhibited BTSC proliferation without inducing cell death. Quantitative functional 2-D and 3-D invasion assays showed that treatment with Sema3A resulted in increased invasion. Using shRNA lentiviruses, knockdown of either NRP1 or PlxnA1 receptors abrogated Sema3A antiproliferative and pro-invasive effects. Interestingly, loss of the receptors mimicked Sema3A effects, inhibiting BTSC proliferation and driving invasion. Furthermore, in vivo studies comparing tumor growth of knockdown and control infected BTSCs implanted into the flanks of nude mice confirmed the decrease in proliferation with receptor KD. CONCLUSIONS: These findings demonstrate the importance of Sema3A signaling in GBM BTSC proliferation and invasion, and its potential as a therapeutic target.

Estrogen-related receptor ß activation and isoform shifting by cdc2-like kinase inhibition restricts migration and intracranial tumor growth in glioblastoma.

Glioblastoma (GBM; grade 4 glioma) is a highly aggressive and incurable tumor. GBM has recently been characterized as highly dependent on alternative splicing, a critical driver of tumor heterogeneity and plasticity. Estrogen-related receptor ß (ERR-ß) is an orphan nuclear receptor expressed in the brain, where alternative splicing of the 3' end of the pre-mRNA leads to the production of 3 validated ERR-ß protein products: ERR-ß short form (ERR-ßsf), ERR-ß2, and ERR-ß exon 10 deleted. Our prior studies have shown the ERR-ß2 isoform to play a role in G2/M cell cycle arrest and induction of apoptosis, in contrast to the function of the shorter ERR-ßsf isoform in senescence and G1 cell cycle arrest. In this study, we sought to better define the role of the proapoptotic ERR-ß2 isoform in GBM. We show that the ERR-ß2 isoform is located not only in the nucleus but also in the cytoplasm. ERR-ß2 suppresses GBM cell migration and interacts with the actin nucleation-promoting factor cortactin, and an ERR-ß agonist is able to remodel the actin cytoskeleton and similarly suppress GBM cell migration. We further show that inhibition of the splicing regulatory cdc2-like kinases in combination with an ERR-ß agonist shifts isoform expression in favor of ERR-ß2 and potentiates inhibition of growth and migration in GBM cells and intracranial tumors.-Tiek, D. M., Khatib, S. A., Trepicchio, C. J., Heckler, M. M., Divekar, S. D., Sarkaria, J. N., Glasgow, E., Riggins, R. B. Estrogen-related receptor ß activation and isoform shifting by cdc2-like kinase inhibition restricts migration and intracranial tumor growth in glioblastoma.

Myosin-1E interacts with FAK proline-rich region 1 to induce fibronectin-type matrix.

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase involved in development and human disease, including cancer. It is currently thought that the four-point one, ezrin, radixin, moesin (FERM)-kinase domain linker, which contains autophosphorylation site tyrosine (Y) 397, is not required for in vivo FAK function until late midgestation. Here, we directly tested this hypothesis by generating mice with FAK Y397-to-phenylalanine (F) mutations in the germline. We found that Y397F embryos exhibited reduced mesodermal fibronectin (FN) and osteopontin expression and died during mesoderm development akin to FAK kinase-dead mice. We identified myosin-1E (MYO1E), an actin-dependent molecular motor, to interact directly with the FAK FERM-kinase linker and induce FAK kinase activity and Y397 phosphorylation. Active FAK in turn accumulated in the nucleus where it led to the expression of osteopontin and other FN-type matrix in both mouse embryonic fibroblasts and human melanoma. Our data support a model in which FAK Y397 autophosphorylation is required for FAK function in vivo and is positively regulated by MYO1E.

Evaluating infusate parameters for direct drug delivery to the brainstem: a comparative study of convection-enhanced delivery versus osmotic pump delivery.

OBJECTIVE: Convection-enhanced delivery (CED) and osmotic pump delivery both have been promoted as promising techniques to deliver drugs to pediatric diffuse intrinsic pontine gliomas (DIPGs). Correspondingly, the aim of this study was to understand how infusate molecular weight (MW), duration of delivery, and mechanism of delivery (CED or osmotic pump) affect volume of distribution (Vd) in the brainstem, to better inform drug selection and delivery in future DIPG investigations. METHODS: A series of in vivo experiments were conducted using rat models. CED and osmotic pump delivery systems were surgically implanted in the brainstem, and different MW fluorescent dextran beads were infused either once (acute) or daily for 5 days (chronic) in a volume infused (Vi). Brainstems were harvested after the last infusion, and Vd was quantified using serial sectioning and fluorescence imaging. RESULTS: Fluorescence imaging showed infusate uptake within the brainstem for both systems without complication. A significant inverse relationship was observed between infusate MW and Vd in all settings, which was distinctly exponential in nature in the setting of acute delivery across the 570-Da to 150-kDa range. Chronic duration and CED technique resulted in significantly greater Vd compared to acute duration or osmotic pump delivery, respectively. When accounting for Vi, acute infusion yielded significantly greater Vd/Vi than chronic infusion. The distribution in CED versus osmotic pump delivery was significantly affected by infusate MW at higher weights. CONCLUSIONS: Here the authors demonstrate that infusate MW, duration of infusion, and infusion mechanism all impact the Vd of an infused agent and should be considered when selecting drugs and infusion parameters for novel investigations to treat DIPGs.

Inhibition of phosphatidylinositol 3-kinase by PX-866 suppresses temozolomide-induced autophagy and promotes apoptosis in glioblastoma cells.

BACKGROUND: Temozolomide (TMZ) is the most commonly used chemotherapeutic agent used to treat glioblastoma (GBM), which causes significant DNA damage to highly proliferative cells. Our observations have added to accumulating evidence that TMZ induces stress-responsive cellular programs known to promote cell survival, including autophagy. As such, targeting these survival pathways may represent new vulnerabilities of GBM after treatment with TMZ. METHODS: Using the T98G human glioma cell line, we assessed the molecular signaling associated with TMZ treatment, the cellular consequences of using the pan-PI3K inhibitor PX-866, and performed clonogenic assays to determine the effect sequential treatment of TMZ and PX-866 had on colony formation. Additionally, we also use subcutaneous GBM patient derived xenograft (PDX) tumors to show relative LC3 protein expression and correlations between survival pathways and molecular markers which dictate clinical responsiveness to TMZ. RESULTS: Here, we report that TMZ can induce autophagic flux in T98G glioma cells. GBM patient-derived xenograft (PDX) tumors treated with TMZ also display an increase in the autophagosome marker LC3 II. Additionally, O6-methylguanine-DNA-methyltransferase (MGMT) expression correlates with PI3K/AKT activity, suggesting that patients with inherent resistance to TMZ (MGMT-high) would benefit from PI3K/AKT inhibitors in addition to TMZ. Accordingly, we have identified that the blood-brain barrier (BBB) penetrant pan-PI3K inhibitor, PX-866, is an early-stage inhibitor of autophagic flux, while maintaining its ability to inhibit PI3K/AKT signaling in glioma cells. Lastly, due to the induction of autophagic flux by TMZ, we provide evidence for sequential treatment of TMZ followed by PX-866, rather than combined co-treatment, as a means to shut down autophagy-induced survival in GBM cells and to enhance apoptosis. CONCLUSIONS: The understanding of how TMZ induces survival pathways, such as autophagy, may offer new therapeutic vulnerabilities and opportunities to use sequential inhibition of alternate pro-survival pathways that regulate autophagy. As such, identification of additional ways to inhibit TMZ-induced autophagy could enhance the efficacy of TMZ.

Automatic 3D Nonlinear Registration of Mass Spectrometry Imaging and Magnetic Resonance Imaging Data.

Multimodal integration between mass spectrometry imaging (MSI) and radiology-established modalities such as magnetic resonance imaging (MRI) would allow the investigations of key questions in complex biological systems such as the central nervous system. Such integration would provide complementary multiscale data to bridge the gap between molecular and anatomical phenotypes, potentially revealing new insights into molecular mechanisms underlying anatomical pathologies presented on MRI. Automatic coregistration between 3D MSI/MRI is a computationally challenging process due to dimensional complexity, MSI data sparsity, lack of direct spatial-correspondences, and nonlinear tissue deformation. Here, we present a new computational approach based on stochastic neighbor embedding to nonlinearly align 3D MSI to MRI data, identify and reconstruct biologically relevant molecular patterns in 3D, and fuse the MSI datacube to the MRI space. We demonstrate our method using multimodal high-spectral resolution matrix-assisted laser desorption ionization (MALDI) 9.4 T MSI and 7 T in vivo MRI data, acquired from a patient-derived, xenograft mouse brain model of glioblastoma following administration of the EGFR inhibitor drug of Erlotinib. Results show the distribution of some identified molecular ions of the EGFR inhibitor erlotinib, a phosphatidylcholine lipid, and cholesterol, which were reconstructed in 3D and mapped to the MRI space. The registration quality was evaluated on two normal mouse brains using the Dice coefficient for the regions of brainstem, hippocampus, and cortex. The method is generic and can therefore be applied to hyperspectral images from different mass spectrometers and integrated with other established in vivo imaging modalities such as computed tomography (CT) and positron emission tomography (PET).

Brain Distribution and Active Efflux of Three panRAF Inhibitors: Considerations in the Treatment of Melanoma Brain Metastases.

Targeted inhibition of RAF and MEK by molecularly targeted agents has been employed as a strategy to block aberrant mitogen-activated protein kinase (MAPK) signaling in melanoma. While the use of BRAF and MEK inhibitors, either as a single agent or in combination, improved efficacy in BRAF-mutant melanoma, initial responses are often followed by relapse due to acquired resistance. Moreover, some BRAF inhibitors are associated with paradoxical activation of the MAPK pathway, causing the development of secondary malignancies. The use of panRAF inhibitors, i.e., those that target all isoforms of RAF, may overcome paradoxical activation and resistance. The purpose of this study was to perform a quantitative assessment and evaluation of the influence of efflux mechanisms at the blood-brain barrier (BBB), in particular, Abcb1/P-glycoprotein (P-gp) and Abcg2/breast cancer resistance protein (Bcrp), on the brain distribution of three panRAF inhibitors: CCT196969 [1-(3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)phenyl)urea], LY3009120 1-(3,3-Dimethylbutyl)-3-(2-fluoro-4-methyl-5-(7-methyl-2-(methylamino)pyrido(2,3-d)pyrimidin-6-yl)phenyl)urea, and MLN2480 [4-pyrimidinecarboxamide, 6-amino-5-chloro-N-[(1R)-1-[5-[[[5-chloro-4-(trifluoromethyl)-2-pyridinyl]amino]carbonyl]-2-thiazolyl]ethyl]-]. In vitro studies using transfected Madin-Darby canine kidney II cells indicate that only LY3009120 and MLN2480 are substrates of Bcrp, and none of the three inhibitors are substrates of P-gp. The three panRAF inhibitors show high nonspecific binding in brain and plasma. In vivo studies in mice show that the brain distribution of CCT196969, LY3009120, and MLN2480 is limited, and is enhanced in transgenic mice lacking P-gp and Bcrp. While MLN2480 has a higher brain distribution, LY3009120 exhibits superior in vitro efficacy in patient-derived melanoma cell lines. The delivery of a drug to the site of action residing behind a functionally intact BBB, along with drug potency against the target, collectively play a critical role in determining in vivo efficacy outcomes.