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von Willebrand factor (VWF) plays a key role in normal hemostasis, and deficiencies of VWF lead to clinically significant bleeding. We sought to identify novel modifiers of VWF levels in endothelial colony-forming cells (ECFCs) using single-cell RNA sequencing (scRNA-seq). ECFCs were isolated from patients with low VWF levels (plasma VWF antigen levels between 30 and 50 IU/dL) and from healthy controls. Human umbilical vein endothelial cells were used as an additional control cell line. Cells were characterized for their Weibel Palade body (WPB) content and VWF release. scRNA-seq of all cell lines was performed to evaluate for gene expression heterogeneity and for candidate modifiers of VWF regulation. Candidate modifiers identified by scRNA-seq were further characterized with small-interfering RNA (siRNA) experiments to evaluate for effects on VWF. We observed that ECFCs derived from patients with low VWF demonstrated alterations in baseline WPB metrics and exhibit impaired VWF release. scRNA-seq analyses of these endothelial cells revealed overall decreased VWF transcription, mosaicism of VWF expression, and genes that are differentially expressed in low VWF ECFCs and control endothelial cells (control ECs). An siRNA screen of potential VWF modifiers provided further evidence of regulatory candidates, and 1 such candidate, FLI1, alters the transcriptional activity of VWF. In conclusion, ECFCs from individuals with low VWF demonstrate alterations in their baseline VWF packaging and release compared with control ECs. scRNA-seq revealed alterations in VWF transcription, and siRNA screening identified multiple candidate regulators of VWF.

Diffuse midline gliomas (DMGs) are incurable pediatric tumors with extraordinarily limited treatment options. Decades of clinical trials combining conventional chemotherapies with radiation therapy have failed to improve these outcomes, demonstrating the need to identify and validate druggable biologic targets within this disease. NTRK1/2/3fusions are found in a broad range of pediatric cancers, including high-grade gliomas and a subset of DMGs. Phase 1/2 studies of TRK inhibitors have demonstrated good tolerability, effective CNS penetration, and promising objective responses across all patients with TRK fusion-positive cancers, but their use has not been explored in TRK fusion-positive DMG. Here, we report 3 cases of NTRK fusions co-occurring within H3K27M-positive pontine diffuse midline gliomas. We employ a combination of single-cell and bulk transcriptome sequencing from TRK fusion-positive DMG to describe the phenotypic consequences of this co-occurring alteration. We then use ex vivo short-culture assays to evaluate the potential response to TRK inhibition in this disease. Together, these data highlight the importance of routine molecular characterization of these highly aggressive tumors and identify a small subset of patients that may benefit from currently available targeted therapies.

High-grade gliomas (HGG) afflict both children and adults and respond poorly to current therapies. Epigenetic regulators have a role in gliomagenesis, but a broad, functional investigation of the impact and role of specific epigenetic targets has not been undertaken. Using a two-step, in vitro/in vivo epigenomic shRNA inhibition screen, we determine the chromatin remodeler BPTF to be a key regulator of adult HGG growth. We then demonstrate that BPTFknockdown decreases HGG growth in multiple pediatric HGG models as well. BPTF appears to regulate tumor growth through cell self-renewal maintenance, and BPTFknockdown leads these glial tumors toward more neuronal characteristics. BPTF’s impact on growth is mediated through positive effects on expression of MYCand MYC pathway targets. HDAC inhibitors synergize with BPTFknockdown against HGG growth. BPTF inhibition is a promising strategy to combat HGG through epigenetic regulation of the MYC oncogenic pathway.

The current treatment for medulloblastoma includes surgical resection, radiation, and cytotoxic chemotherapy. Although this approach has improved survival rates, the high doses of chemotherapy required for clinical efficacy often result in lasting neurocognitive defects and other adverse events. Therefore, the development of chemosensitizing agents that allow dose reductions of cytotoxic agents, limiting their adverse effects but maintaining their clinical efficacy, would be an attractive approach to treat medulloblastoma. We previously identified WEE1 kinase as a new molecular target for medulloblastoma from an integrated genomic analysis of gene expression and a kinome-wide siRNA screen of medulloblastoma cells and tissue. In addition, we demonstrated that WEE1 prevents DNA damage-induced cell death by cisplatin and that the WEE1 inhibitor AZD1775 displays synergistic activity with cisplatin. AZD1775 was developed as a WEE1 inhibitor from an initial hit from a high-throughput screen. However, given the lack of structure–activity data for AZD1775, we developed a small series of analogs to determine the requirements for WEE1 inhibition and further examine the effects of WEE1 inhibition in medulloblastoma. Interestingly, the compounds that inhibited WEE1 in the same nanomolar range as AZD1775 had significantly reduced single-agent cytotoxicity compared with AZD1775 and displayed synergistic activity with cisplatin in medulloblastoma cells. The potent cytotoxicity of AZD1775, unrelated to WEE1 inhibition, may result in dose-limiting toxicities and exacerbate adverse effects; therefore, WEE1 inhibitors that demonstrate low cytotoxicity could be dosed at higher concentrations to chemosensitize the tumor and potentiate the effect of DNA-damaging agents such as cisplatin.

Von Willebrand Factor (VWF) plays a key role in normal hemostasis and deficiencies of VWF lead to clinically significant bleeding. We sought to identify novel modifiers of VWF levels in endothelial colony forming cells (ECFCs) using single cell RNA sequencing (scRNA-seq).ECFCs were isolated from patients with Low VWF levels (plasma VWF antigen levels between 30-50 IU/dL) and from healthy controls. Human umbilical vein endothelial cells were used as an additional control cell line. Cells were characterized for their Weibel Palade body (WPB) content and VWF release. scRNA-seq ofall cell lines was performed to evaluate for gene expression heterogeneity and forcandidate modifiers of VWF regulation. Candidate modifiers identified by scRNA-seq were further characterized with siRNA experiments to evaluate for effects on VWF. We observed that ECFCs derived from patients with Low VWFdemonstratedalterations in baseline WPB metrics and exhibit impaired VWF release. scRNA-seq analyses of these endothelial cells revealed overall decreased VWFtranscription, mosaicism of VWFexpression, and genes that are differentially expressed in Low VWF ECFCsand control endothelial cells (control ECs). A siRNA screen of potential VWF modifiers provided further evidence of regulatory candidates, and one such candidate, FLI1, alters the transcriptional activity of VWF.In conclusion, ECFCs from Low VWF individuals demonstrate alterations in their baseline VWF packaging and release as compared to control ECs.scRNA-seq revealed alterations in VWFtranscription andsiRNA screening identified multiple candidate regulators of VWF.

This study investigates the hypothesis that Mn-superoxide dismutase (MnSOD) influences cancer cell radiosensitivity by regulating the G2-checkpoint pathway. Human oral squamous carcinoma cells (SCC25) stably overexpressing MnSOD were irradiated (6 Gy) and assayed for cell survival, cell-cycle phase distributions, and bromodeoxyuridine (BrdU) pulse-chase flow-cytometric measurements of cell-cycle phase transits. Electron paramagnetic resonance (EPR) spectroscopy was used to measure steady-state levels of oxygencentered free radicals. Glutathione and glutathione disulfide levels were used as indicators of changes in the intracellular redox state. MnSOD overexpression increased radioresistance threefold to fourfold; this increase was associated with twofold to threefold increases in radiation-induced G2 accumulation. BrdU pulsechase and flow-cytometric measurements of the percentage of G1 and relative movement showed no significant changes in G1 and S transits; however, the percentage of G2 cells and BrdU-positive cells showed delayed G2+M transits in MnSOD-overexpressing irradiated cells. The steady-state levels of oxygen-centered free radicals were not significantly different in vector compared with MnSOD-overexpressing cells, suggesting that the free radical generation is essentially similar. MnSOD overexpression did prevent radiation-induced decreases in total glutathione content, which correlated with radioresistance and enhanced G2 accumulation. These results support the hypothesis that a "metabolic redox-response" to IR exposure regulates radiosensitivity by altering radiation-induced G2 accumulation.

Cells oxidize molecules to generate energy and to make the materials to build and support the structures and functions needed for life. However, unwanted oxidations can damage these same structures and impair function. Lipids (the lipids in membranes and lipoproteins) are targets of unwanted oxidations. The primary mechanism of these oxidations is free radical-mediated chain reactions. Here we provide an overview of how electron paramagnetic resonance (EPR) can be used to detect the free radicals formed during lipid peroxidation. Although direct detection of lipid-derived radicals has been accomplished, the approach is not feasible for detecting these radicals in cells. Spin trapping with α-(4-pyridyl-1-oxide)-N-tert-butylnitrone and 5,5-dimethyl-pyrroline-1-oxide has provided the most information on cellular lipid peroxidation. We present some considerations for successful detection of lipid radicals by EPR.

Matrix metalloproteinases (MMPs) participate in cell migration and remodeling processes by affecting the extracellular matrix. MMP-2 is thought to be involved in cancer cell invasiveness. It has been proposed that the activity of MMP-2 can be modulated by intracellular reactive oxygen species (ROS)/reactive nitrogen species. We hypothesized that manganese superoxide dismutase (MnSOD) could mediate MMP-2 activity by changing the intracellular ROS level and that nitric oxide (⋅NO) may be involved in this process. Human breast cancer MCF-7 cells were stably transfected with plasmids containing MnSOD cDNA. A 2–30-fold increase of MnSOD protein and activity was observed in four clones. Our data demonstrated that overexpression of MnSOD stimulated the activation of MMP-2 with a corresponding elevation of ROS. A decrease in ROS by ebselen, a glutathione peroxidase mimetic, or by transduction of adenovirus containing human catalase or glutathione peroxidase cDNA abolished the effect of MnSOD on MMP-2 activation. Treatment of MCF-7 cells with antimycin A or rotenone increased intracellular ROS production and MMP-2 activation simultaneously. Our data also showed a suppression of endothelial nitric-oxide synthase expression that was accompanied by decreased ⋅NO production in MnSOD-overexpressing cells. However, the changes in endothelial nitric-oxide synthase and⋅NO did not correlate with the MnSOD activity. Corresponding changes of MMP-2 activity after the addition of a NOS inhibitor (NG-amino-l-arginine) or a⋅NO donor ((Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate) to the cells suggested the possibility that ⋅NO may be involved in the MnSOD-mediated MMP-2 activation pathway. These results indicate that MnSOD induces MMP-2 activity by regulation of intracellular ROS and imply that signaling pathways involving ⋅NO may also be involved in the MnSOD mediation of MMP-2 activity.

AbstractWe report here the synthesis, crystal structure and Electron Paramagnetic Resonance (EPR) studies of the compound bis[1,3-di(p-n-octylphenyl)propane-1,3-dionato]oxovanadium(IV) abbreviated as C8(VO)C8. The molecular crystalline properties of this compound are dealt with in this paper. The compound crystallizes in the triclinic, P1space group with a = 11.956(4), b = 15.802(5), c = 16.293(4) Å, α = 103.22(2), β = 110.61(2), and γ = 90.88(3); Z=2. The two vanadium centers are crystallographically equivalent and the shortest V-V bond distance is 9.5803 Å. The hyperfine splitting pattern in single crystal EPR spectrum clearly shows the presence of weak pair-wise magnetic interaction. The splitting originates in a combination of hyperfine and fine structure caused by dipolar and exchange coupling of nearest-neighboring molecules related by inversion through the unit cell origin. The sign and the value of the exchange coupling constant, J = + 30 G, was determined by simulation of EPR spectra. The principal g and A values obtained are g|= 1.934 and g⊥= 1.982, A|= 192 G and A⊥= 69 G.

MYC-driven medulloblastoma is a major therapeutic challenge due to frequent metastasis and a poor 5-year survival rate. MYC gene amplification results in transcriptional dysregulation, proliferation, and survival of malignant cells. To identify therapeutic targets in MYC-amplified medulloblastoma, we employ a CRISPR-Cas9 essentiality screen targeting 1,140 genes. We identify CDK7 as a mediator of medulloblastoma tumorigenesis. Using chemical inhibitors and genetic depletion, we observe cessation of tumor growth in xenograft mouse models and increases in apoptosis. The results are attributed to repression of a core set of MYC-driven transcriptional programs mediating DNA repair. CDK7 inhibition alters RNA polymerase II (RNA Pol II) and MYC association at DNA repair genes. Blocking CDK7 activity sensitizes cells to ionizing radiation leading to accrual of DNA damage, extending survival and tumor latency in xenograft mouse models. Our studies establish the selective inhibition of MYC-driven medulloblastoma by CDK7 inhibition combined with radiation as a viable therapeutic strategy.

Diffuse intrinsic pontine glioma (DIPG) is an incurable brain tumor of childhood characterized by histone mutations at lysine 27, which results in epigenomic dysregulation. There has been a failure to develop effective treatment for this tumor. Using a combined RNAi and chemical screen targeting epigenomic regulators, we identify the polycomb repressive complex 1 (PRC1) component BMI1 as a critical factor for DIPG tumor maintenance in vivo. BMI1 chromatin occupancy is enriched at genes associated with differentiation and tumor suppressors in DIPG cells. Inhibition of BMI1 decreases cell self-renewal and attenuates tumor growth due to induction of senescence. Prolonged BMI1 inhibition induces a senescence-associated secretory phenotype, which promotes tumor recurrence. Clearance of senescent cells using BH3 protein mimetics co-operates with BMI1 inhibition to enhance tumor cell killing in vivo.

Ependymoma (EPN) is a brain tumor commonly presenting in childhood that remains fatal in most children. Intra-tumoral cellular heterogeneity in bulk-tumor samples significantly confounds our understanding of EPN biology, impeding development of effective therapy. We, therefore, use single-cell RNA sequencing, histology, and deconvolution to catalog cellular heterogeneity of the major childhood EPN subgroups. Analysis of PFA subgroup EPN reveals evidence of an undifferentiated progenitor subpopulation that either differentiates into subpopulations with ependymal cell characteristics or transitions into a mesenchymal subpopulation. Histological analysis reveals that progenitor and mesenchymal subpopulations co-localize in peri-necrotic zones. In conflict with current classification paradigms, relative PFA subpopulation proportions are shown to determine bulk-tumor-assigned subgroups. We provide an interactive online resource that facilitates exploration of the EPN single-cell dataset. This atlas of EPN cellular heterogeneity increases understanding of EPN biology.

Histone 3 gene mutations are the eponymous drivers in diffuse midline gliomas (DMGs), aggressive pediatric brain cancers for which no curative therapy currently exists. These recurrent oncohistones induce a global loss of repressive H3K27me3 residues and broad epigenetic dysregulation. In order to identify therapeutically targetable dependencies within this disease context, we performed an RNAi screen targeting epigenetic/chromatin-associated genes in patient-derived DMG cultures. This identified AFF4, the scaffold protein of the super elongation complex (SEC), as a molecular dependency in DMG. Interrogation of SEC function demonstrates a key role for maintaining clonogenic potential while promoting self-renewal of tumor stem cells. Small-molecule inhibition of SEC using clinically relevant CDK9 inhibitors restores regulatory RNA polymerase II pausing, promotes cellular differentiation, and leads to potent anti-tumor effect both in vitroand in patient-derived xenograft models. These studies present a rationale for further exploration of SEC inhibition as a promising therapeutic approach to this intractable disease.

The original version of this Article omitted the following from the Acknowledgements: This work was supported by the Luke’s Army Pediatric Cancer Research Fund St. Baldrick’s Scholar Award. This has now been corrected in both the PDF and HTML versions of the Article.

No relevant conflicts of interest to declare.

Background: Von Willebrand disease (VWD) is the most common inherited bleeding disorder with a prevalence ranging from 0.8 to 1.3%.VWD is characterized by incomplete penetrance, variable expressivity with markedly variable VWD antigen levels, even in families with a unifying VWFmutation, suggesting the presence of modifier genes or epigenetic modifications affecting expression of the VWFgene. We had previously diagnosed and characterized an 821-member, multigenerational Amish family in which 121 individuals exhibit a single autosomal-dominant C4120T mutation (R1374C) in the A1 domain of the mature VWF molecule. VWF antigen levels were found to vary significantly, from 9-76 in the individuals exhibiting the C4120T mutation vs 46-483 in the individuals not exhibiting the mutation. To date, very little is known about the epigenetics of VWD and no data exist investigating promoter methylation as a modifier of disease severity. We hypothesized that VWF gene is epigenetically silenced by aberrant DNA methylation resulting in differential expression of VWF protein in patients with VWD. We report our findings suggesting that promoter methylation within the VWFgene may modify VWD severity.