Sarkar, Gobinda

OBJECTIVE A bileaflet aortic valve (BAV) occurs in 1–2% of the population, making it the most common cardiovascular malformation. Patients with BAV have increased risk of thoracic ascending aortic aneurysm (TAA) and dissection compared to those with trileaflet aortic valves (TAV). We have evaluated the gene expression profile of the aorta of patients with BAV and TAV with TAA. METHODS Segment of aneurysmal tissue was collected from 12 patients with BAV (age 43–68) and 19 patients with TAV (age 37–79). RNA was extracted using the Invitrogen RNA extraction Kit. RNA that was of adequate quality by the Agilent Bioanalyzer was used in the Affymetrix protocol for the U133A gene chips probing for over 16,000 genes on each chip. Real‐time RT‐PCR was used to confirm the microarray results for a subset of genes. RESULTS Data filtering (using two different statistical methods) identified significant differences in gene expression between the BAV and TAV groups. The real time RT‐PCR independently confirmed that the expression levels of osteopontin and tenascin were consistently increased in TAA with TAV. CONCLUSIONS The results indicate that many genes are involved in a complicated manner in the pathogenesis of TAA. This study also demonstrates two basic gene expression profiles in the TAA, one characteristic of patients with BAV and the other of those with TAV. Variations in the molecular mechanisms underlying different types of TAA warrant further investigation. (This work was supported by an SS‐50 award to Dr. Thoralf Sundt from the Department of Surgery, Mayo Clinic)

Abstract Purpose: Glioblastoma is the most frequent and lethal primary brain tumor. Development of novel therapies relies on the availability of relevant preclinical models. We have established a panel of 96 glioblastoma patient-derived xenografts (PDX) and undertaken its genomic and phenotypic characterization. Experimental Design: PDXs were established from glioblastoma, IDH-wildtype (n = 93), glioblastoma, IDH-mutant (n = 2), diffuse midline glioma, H3 K27M-mutant (n = 1), and both primary (n = 60) and recurrent (n = 34) tumors. Tumor growth rates, histopathology, and treatment response were characterized. Integrated molecular profiling was performed by whole-exome sequencing (WES, n = 83), RNA-sequencing (n = 68), and genome-wide methylation profiling (n = 76). WES data from 24 patient tumors was compared with derivative models. Results: PDXs recapitulate many key phenotypic and molecular features of patient tumors. Orthotopic PDXs show characteristic tumor morphology and invasion patterns, but largely lack microvascular proliferation and necrosis. PDXs capture common and rare molecular drivers, including alterations of TERT, EGFR, PTEN, TP53, BRAF, and IDH1, most at frequencies comparable with human glioblastoma. However, PDGFRA amplification was absent. RNA-sequencing and genome-wide methylation profiling demonstrated broad representation of glioblastoma molecular subtypes. MGMT promoter methylation correlated with increased survival in response to temozolomide. WES of 24 matched patient tumors showed preservation of most genetic driver alterations, including EGFR amplification. However, in four patient–PDX pairs, driver alterations were gained or lost on engraftment, consistent with clonal selection. Conclusions: Our PDX panel captures the molecular heterogeneity of glioblastoma and recapitulates many salient genetic and phenotypic features. All models and genomic data are openly available to investigators.

Abstract OBJECTIVE The K16ApoE peptide enhances delivery of multiple agents across the blood-brain barrier (BBB). Transgenic mouse models are central to elucidating the underlying pathophysiology of Alzheimer’s Disease (AD) and provide a system for evaluating novel therapeutic strategies. PET imaging plays a central clinical role in diagnosing human cases of AD but has had variable performance in mouse models. We investigated the role of K16ApoE to enhance delivery of a radiolabeled PET imaging tracer, 11C-PiB and assess whether this corresponds to improved PET sensitivity in APP/PS1 transgenic mice. METHODS Brain-delivery of 11C-PiB was accomplished by successive injections of K16ApoE and 11C-PiB. Distribution of 11C-PiB to the brain and heart was quantified via dynamic PET/CT imaging and digital autoradiography. RESULTS K16ApoE increased the brain uptake of 11C-PiB in both wild-type (WT) and APP/PS1 mice. Administration of K16ApoE increased the PET standard uptake value of 11C-PiB at 5 minutes in WT mice from 1.132 to 2.963 (p=0.006) and in APP/PS1 mice from 0.842 to 3.268 (p=0.016). Enhancement peaked at 5 minutes. Binding was reversible with similarly increased kinetics in both WT and APP/PS1 mice. The absolute values were higher in APP/PS1 mice suggesting increased retention. The increased retention in APP/PS1 mice was consistent with specific binding to A-beta plaques as unlabeled PiB reduced 11C-PiB signal retention. CONCLUSION K16ApoE mediates enhancement of 11C-PiB signal in APP/PS1 mice brains with increased PET sensitivity. There is increased uptake kinetics in both WT and APP/PS1 mice with specific A-beta plaque binding in the latter. This enhanced delivery of the PET tracer has implications for development and testing of new hypotheses and the efficacy of novel therapeutic paradigms. K16ApoE has potential for improving delivery of several agents across the BBB. This has implication for delivery of diagnostic and therapeutic agents for neurodegenerative and neuro-oncologic diseases.

Abstract Patients with melanoma have a high risk of developing brain metastasis, which is associated with a dismal prognosis. During early stages of metastasis development, the blood–brain barrier (BBB) is likely intact, which inhibits sufficient drug delivery into the metastatic lesions. We investigated the ability of the peptide, K16ApoE, to permeabilize the BBB for improved treatment with targeted therapies preclinically. Dynamic contrast enhanced MRI (DCE-MRI) was carried out on NOD/SCID mice to study the therapeutic window of peptide-mediated BBB permeabilization. Further, both in vivo and in vitro assays were used to determine K16ApoE toxicity and to obtain mechanistic insight into its action on the BBB. The therapeutic impact of K16ApoE on metastases was evaluated combined with the mitogen-activated protein kinase pathway inhibitor dabrafenib, targeting BRAF mutated melanoma cells, which is otherwise known not to cross the intact BBB. Our results from the DCE-MRI experiments showed effective K16ApoE-mediated BBB permeabilization lasting for up to 1 hour. Mechanistic studies showed a dose-dependent effect of K16ApoE caused by induction of endocytosis. At concentrations above IC50, the peptide additionally showed nonspecific disturbances on plasma membranes. Combined treatment with K16ApoE and dabrafenib reduced the brain metastatic burden in mice and increased animal survival, and PET/CT showed that the peptide also facilitated the delivery of compounds with molecular weights as large as 150 kDa into the brain. To conclude, we demonstrate a transient permeabilization of the BBB, caused by K16ApoE, that facilitates enhanced drug delivery into the brain. This improves the efficacy of drugs that otherwise do not cross the intact BBB.

Abstract BACKGROUND: Melanoma patients have a high risk of developing brain metastases, which is associated with a poor prognosis. The blood-brain barrier (BBB) inhibits sufficient drug delivery into metastatic lesions. We investigated the ability of a synthetic peptide (K16ApoE) to permeabilize the BBB for more effective drug treatment. METHODS: DCE-MRI was performed to study the therapeutic window of BBB opening facilitated by K16ApoE. In vivoand in vitroassays were used to determine K16ApoE toxicity and also to obtain mechanistic insight into its action on the BBB. The therapeutic impact of K16ApoE on melanoma metastases was determined together with dabrafenib, which is otherwise known not to cross an intact BBB. RESULTS: DCE-MRI exhibited an effective K16ApoE-mediated BBB opening for up to 1h. Mechanistic studies displayed a dose-dependent effect of K16ApoE caused by induction of endocytosis. At higher concentrations, the peptide also showed unspecific disturbances on plasma membranes. Combined treatment with K16ApoE and dabrafenib reduced the brain metastatic burden in mice compared to dabrafenib. We also showed by PET/CT that the peptide facilitated the delivery of compounds up to 150 kDa into the brain. CONCLUSIONS: We demonstrate a transient opening of the BBB, caused by K16ApoE, that facilitates improved drug-delivery into the brain. This improves the efficacy of drugs that otherwise do not cross the intact BBB.

Abstract Introduction: Patients with brain metastases await a dismal prognosis. Regardless of the continuous progress in drug development, a major problem is the delivery of drugs across the blood brain barrier (BBB) and into the metastatic neoplasms. The BBB excludes almost all compounds, in particular highly charged, hydrophilic or large compounds, and most of the current chemotherapeutic agents are thus unable to penetrate the BBB. Varying strategies to transiently open the BBB have been studied previously. Here, we describe a peptide transporter comprising 16 lysine residues and 20 amino acid residues corresponding to the low density lipoprotein receptor (LDLR) binding domain of apolipoprotein E (ApoE). We show that the peptide (K16ApoE) is able to transiently open the BBB for drug-delivery into experimental brain metastases. Experimental procedures: A systemic study of the ability of the peptide to open the BBB was conducted by dynamic contrast enhanced magnetic resonance imaging (DCE MRI) in nonobese diabetic/severe combined (nod/scid) mice. The BBB permeability was studied after administering 200 μg of the peptide intravenously. Further, cellular effects after treatment with the peptide was investigated in vitro using confocal microscopy, flow cytometry and impedance experiments. The biodistribution of the peptide was studied in blood plasma and several organs using 125I labeled K16ApoE. Finally, a treatment study was initiated, treating the animals with the peptide in combination with the B-RAF inhibitor Dabrafenib, only Dabrafenib or vehicle. Summary: After injecting the K16ApoE peptide into the mice, a transient opening of the BBB for up to 4 hours was clearly demonstrated by DCE-MRI. Microscopy showed that the peptide disrupted brain endothelial cell monolayers, reducing the barrier properties of the cells. The impedance experiments displayed that the permeability through endothelial cell barriers was increased after treatment with K16ApoE, and a dose-dependent cell death pattern was observed at higher concentrations of K16ApoE.The peptide did not affect endothelial cell tight junctions. The biodistribution study showed that the peptide was eliminated from blood plasma in less than five minutes through the kidneys. The treatment study displayed that the group of animals receiving K16ApoE followed by Dabrafenib had smaller tumor volumes than the other two animal groups. Conclusions: We have shown that the peptide opens the BBB and facilitates a therapeutic window of 4 hours. The peptide did in combination with Dabrafenib decrease the number of experimental brain metastases in our studies. Thus, the current strategy could also have the potential to improve the treatment of patients with brain metastatic disease. Citation Format: Synnøve Nymark Aasen, Heidi Espedal, Olivier Keunen, Christopher Florian Holte, Habib Baghirov, Rolf Bjerkvig, Tine Veronica Karlsen, Olav Tenstad, Dag Erlend Olberg, Gobinda Sarkar, Robert B Jenkins, Frits Thorsen. The peptide transporter K16ApoE increases drug delivery across the blood brain barrier in an experimental animal model of melanoma brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2865. doi:10.1158/1538-7445.AM2017-2865

ABSTRACTOsteosarcoma is a bone tumor that mainly affects children and adolescents. Although its pathogenesis is still not fully understood, activation of Wnt signaling has been implicated in the development and metastasis of osteosarcoma. In this report, we have investigated the effect of the anti‐tumor compound, 2‐methoxyestradiol (2‐ME) on Wnt antagonist frizzled‐related protein b (Frzb), also known as secreted frizzled‐related protein (sFRP)3 in human osteosarcoma (MG63) cells. Our results show that 2‐ME treatment induces Frzb gene promoter activity, and increases Frzb mRNA and protein levels in osteosarcoma cells. In addition, 2‐ME treatment regulates downstream Wnt signaling, increasing the cytoplasmic levels of β‐catenin, and blocking β‐catenin‐mediated Wnt activation in osteosarcoma cells. 2‐ME‐mediated induction of Frzb protein expression is specific to osteosarcoma cells, as it does not affect Frzb expression in normal primary human osteoblasts. Furthermore, 2‐ME‐induced apoptosis and autophagy are blocked in osteosarcoma cells transfected with Frzb siRNAs. Taken together, these studies demonstrate that Frzb protein plays an important role in 2‐ME‐mediated anti‐tumor mechanisms in osteosarcoma cells. J. Cell. Biochem. 118: 1497–1504, 2017. © 2016 Wiley Periodicals, Inc.