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Supplementary Video 5 shows a confocal time-lapse series, showing MDCK cells treated with 40 ug of K16ApoE, immediately after labeling with Calcein-AM (green), which indicates live cells, and Ethidium homodimer-1 (red), indicating dead cells.

Supplementary Video 2 shows sonographic visualization of the axial position of the heart of a NOD/SCID mouse injected with 1000 ug K16ApoE intravenously.

Supplementary Video 4 shows a confocal time-lapse series, showing MDCK cells treated with 20 ug of K16ApoE, immediately after labeling with Calcein-AM (green), which indicates live cells, and Ethidium homodimer-1 (red), indicating dead cells.

Supplementary Video 1 shows sonographic visualization of the axial position of the heart of a NOD/SCID mouse injected with 9 mg/mL NaCl intravenously.

Supplementary Video 3 shows a confocal time-lapse series, showing a control sample of MDCK cells labeled with Calcein-AM (green), which indicates live cells, and Ethidium homodimer-1 (red), indicating dead cells.

Supplementary Video 6 shows a confocal time-lapse series, showing MDCK cells treated with 80 ug of K16ApoE, immediately after labeling with Calcein-AM (green), which indicates live cells, and Ethidium homodimer-1 (red), indicating dead cells.

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.

Supplementary Materials shows supplementary methods, Supplementary Figures S1-S9 with legends and Supplementary Table 1. Supplementary Table 1: Mean Ve, Kep and AUC after DCE-MRI; Supplementary Figure S1: In vivo study design; Supplementary Figure S2: Blood smear from mice subjected to 1,000 µg K16ApoE; Supplementary Figure S3: In vitro cell viability: Supplementary Figure S4: Mechanistic studies of the in vitro effects of K16ApoE; Supplementary Figure S5: In vitro BBB model; Supplementary Figure S6: Biodistribution of 125I-K16ApoE; Supplementary Figure S7: Histopathological evaluation of tissue samples after long- and short-term treatment of NOD/SCID mice with the peptide; Supplementary Figure S8: In vivo treatment control experiment; Supplementary Figure S9: Mass spectrometry of treated and untreated brain tissue

Genetics of recurrent PDX

Supplementary Table 3 from High WT1 Expression Is Associated with Very Poor Survival of Patients with Osteogenic Sarcoma Metastasis

Supplementary Table 1 from High WT1 Expression Is Associated with Very Poor Survival of Patients with Osteogenic Sarcoma Metastasis

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.

Figures S1-S8, Supplementary Methods, Supplementary Bibliography

Supplementary Table 3 from Sleeping Beauty–Mediated Somatic Mutagenesis Implicates CSF1 in the Formation of High-Grade Astrocytomas

Supplementary Figure 3 from Sleeping Beauty–Mediated Somatic Mutagenesis Implicates CSF1 in the Formation of High-Grade Astrocytomas

Supplementary Figure 1 from Sleeping Beauty–Mediated Somatic Mutagenesis Implicates CSF1 in the Formation of High-Grade Astrocytomas

Supplementary Table 4 from Sleeping Beauty–Mediated Somatic Mutagenesis Implicates CSF1 in the Formation of High-Grade Astrocytomas

Supplementary Figure 4 from Sleeping Beauty–Mediated Somatic Mutagenesis Implicates CSF1 in the Formation of High-Grade Astrocytomas

Supplementary Table 1 from Sleeping Beauty–Mediated Somatic Mutagenesis Implicates CSF1 in the Formation of High-Grade Astrocytomas

Supplementary Table 2 from Sleeping Beauty–Mediated Somatic Mutagenesis Implicates CSF1 in the Formation of High-Grade Astrocytomas

Supplementary Table 5 from Sleeping Beauty–Mediated Somatic Mutagenesis Implicates CSF1 in the Formation of High-Grade Astrocytomas

Supplementary Figure 2 from Sleeping Beauty–Mediated Somatic Mutagenesis Implicates CSF1 in the Formation of High-Grade Astrocytomas

The CRISPR-Cas9 system has enabled researchers to precisely modify/edit the sequence of a genome. A typical editing experiment consists of two steps: (i) editing cultured cells; (ii) cell cloning and selection of clones with and without intended edit, presumed to be isogenic. The application of CRISPR-Cas9 system may result in off-target edits, while cloning will reveal culture-acquired mutations. We analyzed the extent of the former and the latter by whole genome sequencing in three experiments involving separate genomic loci and conducted by three independent laboratories. In all experiments we hardly found any off-target edits, while detecting hundreds to thousands of single nucleotide mutations unique to each clone after relatively short culture of 10-20 passages. Notably, clones also differed in copy number alterations that were several kb to several mb in size and represented the largest source of genomic divergence among clones. We suggest that screening of clones for mutations and copy number alterations acquired in culture is a necessary step to allow correct interpretation of DNA editing experiments. Furthermore, since culture associated mutations are inevitable, we propose that experiments involving derivation of clonal lines should compare a mix of multiple unedited lines and a mix of multiple edited lines.

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.

Accumulation of amyloid beta (Aβ) peptides in the cerebral vasculature, referred to as cerebral amyloid angiopathy (CAA), is widely observed in Alzheimer's disease (AD) brain and was shown to accelerate cognitive decline. There is no effective method for detecting cerebrovascular amyloid (CVA) and treat CAA. The targeted nanoparticles developed in this study effectively migrated from the blood flow to the vascular endothelium as determined by using quartz crystal microbalance with dissipation monitoring (QCM-D) technology. We also improved the stability, and blood–brain barrier (BBB) transcytosis of targeted nanoparticles by coating them with a cationic BBB penetrating peptide (K16ApoE). The K16ApoE-Targeted nanoparticles demonstrated specific targeting of vasculotropic DutchAβ40 peptide accumulated in the cerebral vasculature. Moreover, K16ApoE-Targeted nanoparticles demonstrated significantly greater uptake into brain and provided specific MRI contrast to detect brain amyloid plaques.

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.

Osteosarcoma 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.

Aberrant or constitutive activation of nuclear factor kappa B (NF-κB) contributes to various human inflammatory diseases and malignancies via the upregulation of genes involved in cell proliferation, survival, angiogenesis, inflammation, and metastasis. Thus, inhibition of NF-κB signaling has potential for therapeutic applications in cancer and inflammatory diseases. We reported previously that Nei-like DNA glycosylase 2 (NEIL2), a mammalian DNA glycosylase, is involved in the preferential repair of oxidized DNA bases from the transcriptionally active sequences via the transcription-coupled base excision repair pathway. We have further shown that Neil2-null mice are highly sensitive to tumor necrosis factor α (TNFα)- and lipopolysaccharide-induced inflammation. Both TNFα and lipopolysaccharide are potent activators of NF-κB. However, the underlying mechanism of NEIL2's role in the NF-κB–mediated inflammation remains elusive. Here, we have documented a noncanonical function of NEIL2 and demonstrated that the expression of genes, such as Cxcl1, Cxcl2, Cxcl10, Il6, and Tnfα, involved in inflammation and immune cell migration was significantly higher in both mock- and TNFα-treated Neil2-null mice compared with that in the WT mice. NEIL2 blocks NF-κB's binding to target gene promoters by directly interacting with the Rel homology region of RelA and represses proinflammatory gene expression as determined by co-immunoprecipitation, chromatin immunoprecipitation, and electrophoretic mobility-shift assays. Remarkably, intrapulmonary administration of purified NEIL2 via a noninvasive nasal route significantly abrogated binding of NF-κB to cognate DNA, leading to decreased expression of proinflammatory genes and neutrophil recruitment in Neil2-null as well as WT mouse lungs. Our findings thus highlight the potential of NEIL2 as a biologic for inflammation-associated human diseases.

Individuals with brain metastasis usually await a poor prognosis. Even with the ongoing development of novel targeted therapies, a major impediment in the treatment of brain metastasis is delivering of drugs across the blood-brain/tumor barrier. Different strategies to accomplish temporary opening of the heterogenous blood-brain barrier have been described. Here, we report on a synthetic peptide, K16ApoE, which is able to open the blood-brain barrier for improved drug-delivery in a non-permanent manner. We carried out extensive dynamic contrast enhanced magnetic resonance imaging experiments to study the ability of the peptide to open the blood-brain barrier in healthy mice. We further performed dynamic positron emission/computer tomography, with F18-labeled albumin and IgG to validate an open blood-brain barrier after co-injecting with K16ApoE. Further, we studied the effects of the peptide on cells and tissue in vitro and in vivo. The biodistribution of the peptide was studied in healthy mice by labeling I-125 to the imidazole ring of histidine in K16ApoE and tracking the peptide in plasma and tissue. We also conducted a treatment study on BRAF(V600E)mutant brain metastases, where the treatment groups received K16ApoE before a BRAF inhibitor was administered, BRAF inhibitor only, K16ApoE only or vehicle. We found a peptide-induced therapeutic window at approximately 30 minutes using dynamic contrast enhanced magnetic resonance imaging. The dynamic positron emission/computer tomography showed cerebral uptake of albumin (∼67 kDa) and IgG (∼150 kDa) in healthy mice with a presumed intact blood-brain barrier. Further, we identified lysis of cells treated with K16ApoE in addition to in part endocytosis-mediated peptide uptake. In vitro blood-brain barrier modeling demonstrated endothelial and astrocyte cell layer regrowth within approximately three and 15 hours, respectively, after treatment with the peptide. We observed a significant treatment effect of co-injecting the peptide with a BRAF inhibitor on BRAF mutated brain metastases.

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.

The blood–brain barrier (BBB) presents a major challenge to effective treatment of neurological disorders, including lysosomal storage diseases (LSDs), which frequently present with life-shortening and untreatable neurodegeneration. There is considerable interest in methods for intravenous delivery of lysosomal proteins across the BBB but for the most part, levels achievable in the brain of mouse models are modest and increased lifespan remains to be demonstrated. In this study, we have investigated delivery across the BBB using a mouse model of late-infantile neuronal ceroid lipofuscinosis (LINCL), a neurodegenerative LSD caused by loss of tripeptidyl peptidase I (TPP1). We have achieved supraphysiological levels of TPP1 throughout the brain of LINCL mice by intravenous (IV) coadministration of recombinant TPP1 with a 36-residue peptide that contains polylysine and a low-density lipoprotein receptor binding sequence from apolipoprotein E. Importantly, IV administration of TPP1 with the peptide significantly reduces brain lysosomal storage, increases lifespan and improves neurological function. This simple “mix and inject” method is immediately applicable towards evaluation of enzyme replacement therapy to the brain in preclinical models and further exploration of its clinical potential is warranted.

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.

© 2017, Springer-Verlag Berlin Heidelberg. The “integrated diagnosis” for infiltrating gliomas in the 2016 revised World Health Organization (WHO) classification of tumors of the central nervous system requires assessment of the tumor for IDH mutations and 1p/19q codeletion. Since TERT promoter mutations and ATRX alterations have been shown to be associated with prognosis, we analyzed whether these tumor markers provide additional prognostic information within each of the five WHO 2016 categories. We used data for 1206 patients from the UCSF Adult Glioma Study, the Mayo Clinic and The Cancer Genome Atlas (TCGA) with infiltrative glioma, grades II-IV for whom tumor status for IDH, 1p/19q codeletion, ATRX, and TERT had been determined. All cases were assigned to one of 5 groups following the WHO 2016 diagnostic criteria based on their morphologic features, and IDH and 1p/19q codeletion status. These groups are: (1) Oligodendroglioma, IDH-mutant and 1p/19q-codeleted; (2) Astrocytoma, IDH-mutant; (3) Glioblastoma, IDH-mutant; (4) Glioblastoma, IDH-wildtype; and (5) Astrocytoma, IDH-wildtype. Within each group, we used univariate and multivariate Cox proportional hazards models to assess associations of overall survival with patient age at diagnosis, grade, and ATRX alteration status and/or TERT promoter mutation status. Among Group 1 IDH-mutant 1p/19q-codeleted oligodendrogliomas, the TERT-WT group had significantly worse overall survival than the TERT-MUT group (HR: 2.72, 95% CI 1.05–7.04, p = 0.04). In both Group 2, IDH-mutant astrocytomas and Group 3, IDH-mutant glioblastomas, neither TERT mutations nor ATRX alterations were significantly associated with survival. Among Group 4, IDH-wildtype glioblastomas, ATRX alterations were associated with favorable outcomes (HR: 0.36, 95% CI 0.17–0.81, p = 0.01). Among Group 5, IDH-wildtype astrocytomas, the TERT-WT group had significantly better overall survival than the TERT-MUT group (HR: 0.48, 95% CI 0.27–0.87), p = 0.02). Thus, we present evidence that in certain WHO 2016 diagnostic groups, testing for TERT promoter mutations or ATRX alterations may provide additional useful prognostic information.

Adult diffuse glioma that harbor IDH mutation and 1p/19q codeletion commonly have CIC mutations; however, the functional and prognostic significance of CIC mutations remains unclear. We hypothesized that when CIC is not mutated, other genes may be inactivated by hypermethylation. Identifying such genes may help to further understand the biology of 1p/19q-codeleted glioma. To test our hypothesis, we utilized a two-stage design. In the discovery stage, 72 IDH-mutated 1p/19q-codeleted tumors were utilized from TCGA (40 CIC mutated, 32 wild-type); all via Illumina 450K methylation array. In the validation stage, 43 IDH-mutated 1p/19q-codeleted tumors were utilized from Mayo Clinic (33 CIC mutated, 10 wild-type); all via Illumina 850K methylation array. All tumors also had TERT promoter mutation. Genome-wide significance (p≤5x10(-8)) was used in the discovery stage to identify differentially methylated probes between CIC mutated vs wild-type tumors. Seven probes reached significance in TCGA; four validated at pCIC. DLL3, located on 19q, had a mean change in beta value between CIC wild-type and mutated tumors of 0.28 (p=1.29x10(-8)) and 0.24 (p=0.0024) in TCGA and Mayo, respectively. ETV1, located on 7p, had a mean change in beta value of 0.30 (p=1.37x10(-8)) and 0.15 (p=0.00011) in TCGA and Mayo, respectively. In the 72 TCGA tumors, we observed an inverse correlation between DLL3 methylation and gene expression amongst CIC mutated and wild-type tumors; -0.42 (p=0.0063) and -0.33 (p=0.069), respectively. There was also an inverse correlation between ETV1 methylation and gene expression amongst CIC mutated and wild-type tumors; -0.68 (pDLL3 and ETV1 was downregulated in CIC wild-type vs mutated tumors (p=0.000027 and 0.0000028, respectively). We confirm that ETV1 has higher gene expression in CIC mutated vs wild-type tumors. Our results suggest that DLL3 expression is inactivated by promoter methylation in CIC wild-type 1p/19q-codeleted oligodendrogliomas; thus, DLL3 may be an alternative target gene on 19q.

Patients with brain metastases usually have a poor prognosis. Regardless of the constant progress in novel drug establishments, a crucial obstacle is the delivery of drugs across the blood brain barrier (BBB) and into the metastatic neoplasms. Different study designs to achieve temporary BBB opening have previously been studied. Here, we describe a synthetic peptide termed K16ApoE, which is able to transiently open the BBB for drug-delivery into experimental brain metastases.We conducted a systemic study of the ability of the peptide to open the BBB by dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) in nonobese diabetic/severe combined (nod/scid) mice. Further, we studied the mechanisms of the peptide using flow cytometry, electron microscopy and dynamic positron emission tomography (PET). The biodistribution of the peptide was studied in blood plasma and organs using I-125 labeled K16ApoE. Finally, a treatment study using the peptide in combination with the B-RAF inhibitor dabrafenib was conducted.An opening of the BBB for up to 60 mins was clearly demonstrated by DCE-MRI. Microscopy showed that the peptide disrupted brain endothelial cell monolayers by reducing the barrier properties of the cells. The treatment study demonstrated that the group of animals receiving K16ApoE followed by dabrafenib had smaller tumor volumes than the other two animal groups.In conclusion, the synthetic K16ApoE peptide in combination with dabrafenib decreased the number of experimental brain metastases. Thus, the current strategy could have the potential to improve the treatment of patients with brain metastatic disease. Citation Format: Synnøve Nymark Aasen, Heidi Espedal, Christopher Florian Holte, Olivier Keunen, Tine Veronica Karlsen, Olav Tenstad, Habib Baghirov, Dag Erlend Olberg, Rolf Bjerkvig, Gobinda Sarkar, Frits Thorsen. The good drug, the bad barrier and the handy peptide: Improved treatment of experimental melanoma brain metastases using a synthetic peptide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-314.

Objective We sought to determine if the gene responsible for bicuspid aortic valve (BAV) in an extended family corresponds to previously reported loci for inherited forms of the disorder. Background Loci at 15q25.1-26 and 9q34 have been reported to be associated with cardiovascular abnormalities involving BAV. Methods Linkage analysis was performed on DNA collected from a large multigenerational family in which BAV disease segregates in an autosomal dominant manner, using microsatellite markers from the regions previously reported to segregate with the phenotype. Results Lod scores were determined for genetic markers near the NOTCH1 gene and for a locus on chromosome 15q25.1-26 previously reported as being linked to BAV. The lod scores were negative or less than 1.0 for all markers tested. Conclusions There is no evidence of linkage of BAV in our pedigree to either the NOTCH1 gene or to the chromosome 15 locus. The disorder in this family appears to be caused by a gene at a novel locus.

BACKGROUND Sensitizing effects of poly-ADP-ribose polymerase inhibitors have been studied in several preclinical models, but a clear understanding of predictive biomarkers is lacking. In this study, in vivo efficacy of veliparib combined with temozolomide (TMZ) was evaluated in a large panel of glioblastoma multiforme (GBM) patient-derived xenografts (PDX) and potential biomarkers were analyzed. METHODS The efficacy of TMZ alone vs TMZ/veliparib was compared in a panel of 28 GBM PDX lines grown as orthotopic xenografts (8-10 mice per group); all tests of statistical significance were two-sided. DNA damage was analyzed by γH2AX immunostaining and promoter methylation of DNA repair gene O6-methylguanine-DNA-methyltransferase (MGMT) by Clinical Laboratory Improvement Amendments-approved methylation-specific polymerase chain reaction. RESULTS The combination of TMZ/veliparib statistically significantly extended survival of GBM models (P < .05 by log-rank) compared with TMZ alone in five of 20 MGMT-hypermethylated lines (average extension in median survival = 87 days, range = 20-150 days), while the combination was ineffective in six MGMT-unmethylated lines. In the MGMT promoter-hypermethylated GBM12 line (median survival with TMZ+veliparib = 189 days, 95% confidence interval [CI] = 59 to 289 days, vs TMZ alone = 98 days, 95% CI = 49 to 210 days, P = .04), the profound TMZ-sensitizing effect of veliparib was lost when MGMT was overexpressed (median survival with TMZ+veliparib = 36 days, 95% CI = 28 to 38 days, vs TMZ alone = 35 days, 95% CI = 32 to 37 days, P = .87), and a similar association was observed in two nearly isogenic GBM28 sublines with an intact vs deleted MGMT locus. In comparing DNA damage signaling after dosing with veliparib/TMZ or TMZ alone, increased phosphorylation of damage-responsive proteins (KAP1, Chk1, Chk2, and H2AX) was observed only in MGMT promoter-hypermethylated lines. CONCLUSION Veliparib statistically significantly enhances (P < .001) the efficacy of TMZ in tumors with MGMT promoter hypermethylation. Based on these data, MGMT promoter hypermethylation is being used as an eligibility criterion for A071102 (NCT02152982), the phase II/III clinical trial evaluating TMZ/veliparib combination in patients with GBM.

BACKGROUND: Recent work indicates that molecularly-defined glioma subgroups have similar clinical presentation and patient prognoses. These molecularly-defined subgroups often incorporate a measure of the tumor's telomere maintenance strategy, defined by mutations in either TERT or ATRX. Somatic mutations in these genes are typically mutually exclusive and there is ongoing debate over which telomere marker should be adopted into neuropathology practice. METHODS: We assessed both TERT promoter mutation status and ATRX mutation status in 1174 infiltrative adult gliomas (Grades II-IV) from the UCSF Adult Glioma Study, The Mayo Clinic, and TCGA using a combination of sequencing and immunohistochemistry. RESULTS: 653 gliomas were TERTmt only (56%), 337 were ATRXmt only (29%), 164 were TERTwt/ATRXwt (14%), and 20 were TERTmt/ATRXmt (2%). Among IDHwt patients, those with TERTwt/ATRXwt tumors had similar overall survival to patients with TERTmt tumors (HR = 0.91, 95%CI = 0.70-1.18, P = 0.47) but significantly worse survival than patients with ATRXmt tumors (HR = 1.88, 95%CI = 1.01-3.51, P = 0.047) after adjusting for age and grade. Among IDHmt patients, those with TERTwt/ATRXwt tumors had significantly worse overall survival than patients with TERTmt tumors (HR = 2.97; 95%CI = 1.43-6.17, P = 0.0035) and moderately worse survival than patients with ATRXmt tumors (HR = 1.61, 95%CI = 0.98-2.67, P = 0.063) after adjusting for age, grade and 1p/19q status. CONCLUSIONS: These data suggest that among patients with IDHwt glioma, ATRX status may be adequate for prognostication, as both TERTmt and TERTwt/ATRXwt patients differed from ATRXmt patients but did not differ from each other. Among IDHmt patients, those with TERTmt tumors, ATRXmt tumors, and TERTwt/ATRXwt tumors all had different survival experiences. This suggests that knowing both ATRX status and TERT status may be important for patients with IDHmt glioma in order to identify the TERTwt/ATRXwt patients.

The proteomic composition of synovial fluid (SF) may hold clues to understanding the molecular basis of arthritis. However, the highly viscous nature and proteomic complexity of SF present a challenge when analyzing results obtained by two-dimensional gel electrophoresis (2D-GE). Several software applications are available for analyzing 2D-GE images. Despite inherent strengths and weaknesses, no comparison between these applications has been reported using SF or any human fluid specimens. We evaluated two common software packages — PDQuest and Progenesis Workstation — for spot detection, matching, and quantitation of 2D-GE images of SF from four patients with arthritic disease. Initially, whole 2D-gel images were analyzed for spot detection, which suggested that PDQuest is more consistent than Progenesis; however, PDQuest appeared to require more user intervention than Progenesis. Subsequently, two small areas (spots well resolved and spots not well resolved) were selected from each gel image, which were analyzed by the software for spot detection, matching, volume, and resolution. These analyses suggest that both tools can quantify well-resolved spots relatively consistently when compared with manual spot detection (the “gold standard”). The “3D viewer” option offered by both tools enables correct spot identification and matching. The strengths and weaknesses of these computer tools can provide guidance in the choice of a particular workstation for identifying biomarkers of arthritis.

Estrogen deficiency impairs fracture healing, while estrogen treatment of ovariectomized rats accelerates fracture healing. To identify genes regulated by estrogen during fracture healing, we evaluated gene expression in calluses from three groups of rats: sham-operated, ovariectomized, and ovariectomized and treated with estrogen. RNA from calluses harvested 10 days after fracture was subjected to DNA microarray-based analysis of 5147 genes. In total, 52 genes were identified whose mRNA expressions were found to be downregulated by ovariectomy but restored with estrogen. Differential mRNA expression of 4 genes (collagen type 2, extracellular superoxide dismutase, urokinase-type plasminogen activator [ u-PA], and ptk-3) was confirmed by reverse transcription polymerase chain reaction. Further, chondrocytes and chondroclasts were positive for u-PA in the junction between cartilage and bone, implying its importance in resorption and remodeling of callus. Identification of these genes is a prerequisite to understanding the mechanism by which estrogen influences the complex metabolic process of fracture repair.

A 48-bp sequence element in intron 1 of the alpha1(II) collagen gene (Col2a1) acts as an enhancer of Col2a1 transcription and contains binding sites for the transcription activator SOX9 and repressor alphaA-crystallin-binding protein 1 (CRYBP1). We hypothesized that abrogating CRYBP1 binding should increase transcription from a promoter associated with the Col2a1 enhancer. We tested this hypothesis by cotransfecting an oligonucleotide (ODN) decoy for CRYBP1 and a luciferase-based reporter vector under the transcriptional control of the Col2a1 promoter linked to the 100-bp enhancer in chondrogenic ATDC5 cells. As a control, we used decoy ODN corresponding to the SOX9 binding site. Transfection with CRYBP1 decoy increased luciferase activity by >2.5-fold in the absence or presence of insulin, whereas SOX9 decoy ODN decreased luciferase activity to about 50% under similar conditions. In addition, the repressive effect of interleukin-1 on Col2a1 transcription through decreasing SOX9 messenger ribonucleic acid (mRNA) expression and increasing CRYBP1 mRNA expression, was counteracted by CRYBP1 decoy ODN. These results provide a rationale for gene therapy in degenerative joint diseases by elevating Col2a1 expression in chondrocytes through oligomimetics of repressor binding sites.

Statins increase bone morphogenetic protein-2 (BMP-2) mRNA expression and subsequently increase new bone formation in vitro. However, the action of statins on the BMP-2 mRNA regulation of cartilage matrix synthesis by chondrocytes is unknown. We evaluated regulation of BMP-2, aggrecan, and type II collagen (COL2) mRNA and (35)S-labeled proteoglycan (PG) synthesis by mevastatin using cultured chondrocytes obtained from articular cartilage of fetal rats. Expression of BMP-2, aggrecan, and COL2 mRNAs were increased in the presence of 2 microM mevastatin on day 2. However, longer (10 day) culture in the presence of the drug decreased the expression of these mRNAs. PG synthesis was increased 3 days after treating the cells with mevastatin, which was also decreased with longer (10 day) mevastatin treatment. These results suggest that mevastatin increases mRNA expression of BMP-2, aggrecan, and COL2 as well as PG synthesis by fetal rat chondrocytes early in the treatment period. We suggest that statins have implications for fracture and cartilage repair.

Knowledge of the cis-acting elements is required for identifying trans-acting splicing factors underlying cartilage-specific alternative splicing of Col2 pre-mRNA. By performing desired deletions in the mouse Col2 pre-mRNA, location of the intronic cis-acting elements was narrowed down to be at or near splice-junction sequences flanking exon 2 of the gene. Introduction: Type II collagen (Col2) pre-mRNA undergoes cartilage-specific alternative splicing involving exon 2 during chondrocyte differentiation. Thus, the trans-acting protein factors that regulate the splicing are associated with the differentiation of chondrocytes. Knowledge of the cognate cis-acting elements is necessary to eventually identify the trans-acting factors. Materials and Methods: To localize the cis-acting sequences, we created several deletions within a minigene containing exon 1 to exon 4 of mouse Col 2 gene and evaluated alternative splicing of the resulting pre-mRNAs in ATDC5 cells, a model of insulin-stimulated chondrocyte differentiation. The first deletion reduced intron 1 from 3799 to 259 bp, the second reduced intron 2 from 1108 to 94 bp, the third combined the above two deletions, and the fourth was derived from the third by removing intron 3 and exon 4. ATDC5 cells harboring these constructs were cultured for up to 21 days with or without insulin. Alternative splicing was evaluated by determining the ratio of Col2B (lacks exon 2) to Col2A (has exon 2) RNAs by reverse transcription-polymerase chain reaction. Results: The deletion in intron 1 had no effect on the alternative splicing while other deletions affected splicing (demonstrated by the presence of splicing intermediates) in cells cultured without insulin or with insulin for 1 week. The splicing intermediates were not seen from any construct when cells were cultured longer (14–21 days) with insulin. Conclusion: These results show that the 259-bp intron 1, the 94-bp intron 2, and exon 2 sequences retained in the fourth construct provide cis-acting signal sufficient for insulin-induced cartilage-specific alternative splicing of Col2 pre-mRNA.

Synovial fluid (SF) is a dynamic reservoir for proteins originating from serum, synovial tissue, and cartilage. The composition of the SF proteome may reflect the pathophysiological conditions affecting the circulatory system and cartilage. Our long-term goal is to identify reliable protein markers for osteoarthritis (OA) in SF. We first evaluated the pattern of SF proteins on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) as a function of protein loading, pH range for isoelectric focusing, and concentration of acrylamide in SDS-PAGE. Removal of albumin and γ- globulins from the samples did not improve the detection of protein spots on 2D-PAGE. The repeatability of protein spot intensity was tested by triplicate 2D-PAGE of a given sample; these experiments showed low intrasample variability (correlation coefficients 0.89–0.95). Differences between multiple samples were tested by comparing the 2D-PAGE of four samples. These experiments showed slightly greater variation between samples (correlation coefficients 0.85–0.93) and a number of differentially expressed proteins. The intensity of 18 protein spots differed more than fivefold, and the intensity of nine protein spots differed more than 100-fold. These results show that 2D-PAGE can be used under standard conditions to screen SF samples and identify a small subset of proteins in SF that are potential markers associated with OA.

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

BACKGROUND: Combinations of anti-cancer medications often yield better outcome than a single medication in the treatment of patients with cancer including patients with brain cancer. However, limitations imposed by the blood-brain barrier (BBB) create serious challenge to deliver even a single drug to the brain, let alone a combination of drugs. We have developed a peptide carrier K16ApoE, which apparently creates transient opening of the BBB. We investigated the ability of the carrier peptide to deliver a three-drug combination (cetuximab, cisplatin and methotrexate) to the brain injected via intravenous route. METHODS: Brain delivery of the drugs was achieved either by separate or combined administration of K16ApoE and a given drug(s) through femoral vein injection. A modification of the method comprised injection of K16ApoE pre-mixed with cetuximab, followed by injection of a drug. Brain uptake of cetuximab was assessed by micro single photon electron computed tomography (microSPECT). Quantification of brain uptake of cisplatin and methotrexate were achieved by atomic absorption spectrometry and LC-MS/MS, respectively. RESULTS: Experiments designed to deliver cetuximab, cisplatin and methotrexate separately show brain-uptake of the drugs was ∼3-fold, ∼40-fold and ∼51-fold greater with K16ApoE than without, amounting to brain uptake of ∼0.5% for cetuximab and ∼1.5% each for cisplatin and methotrexate, respectively, of the injected dose. Experiments designed to deliver cetuximab, cisplatin and methotrexate simultaneously (in a mixture) produced brain uptake of the three drugs at ∼3-fold, ∼18-fold and ∼41-fold greater with K16ApoE than without for cetuximab, cisplatin and methotrexate, respectively, which amounted to brain uptake of∼ 0.5% for cetuximab, ∼0.7% for cisplatin and ∼ 0.5% for methotrexate. SIGNIFICANCE: The results suggest that multiple anti-cancer agents can be simultaneously delivered to the brain via the synthetic carrier peptide K16ApoE. Thus, the method offers a novel therapeutic avenue for intervention against brain cancer.