Glioblastoma vulnerability to neddylation inhibition is dependent on PTEN status, and dysregulation of the cell cycle and DNA replication.

Background: Neddylation (NAE) inhibition, affecting posttranslational protein function and turnover, is a promising therapeutic approach to cancer. We report the cytotoxic vulnerability to NAE inhibitors in a subset of glioblastoma (GBM) preclinical models and identify genetic alterations and biological processes underlying differential response. Methods: GBM DNA sequencing and transcriptomic data were queried for genes associated with response to NAE inhibition; candidates were validated by molecular techniques. Multi-omics and functional assays revealed processes implicated in NAE inhibition response. Results: Transcriptomics and shotgun proteomics depict PTEN signaling, DNA replication, and DNA repair pathways as significant differentiators between sensitive and resistant models. Vulnerability to MLN4924, a NAE inhibitor, is associated with elevated S-phase populations, DNA re-replication, and DNA damage. In a panel of GBM models, loss of WT PTEN is associated with resistance to different NAE inhibitors. A NAE inhibition response gene set could segregate the GBM cell lines that are most resistant to MLN4924. Conclusions: Loss of WT PTEN is associated with non-sensitivity to 3 different compounds that inhibit NAE in GBM. A NAE inhibition response gene set largely consisting of DNA replication genes could segregate GBM cell lines most resistant to NAEi and may be the basis for future development of NAE inhibition signatures of vulnerability and clinical trial enrollment within a precision medicine paradigm.

Droplet Hi-C for Fast and Scalable Profiling of Chromatin Architecture in Single Cells.

Comprehensive analysis of chromatin architecture is crucial for understanding the gene regulatory programs during development and in disease pathogenesis, yet current methods often inadequately address the unique challenges presented by analysis of heterogeneous tissue samples. Here, we introduce Droplet Hi-C, which employs a commercial microfluidic device for high-throughput, single-cell chromatin conformation profiling in droplets. Using Droplet Hi-C, we mapped the chromatin architecture at single-cell resolution from the mouse cortex and analyzed gene regulatory programs in major cortical cell types. Additionally, we used this technique to detect copy number variation (CNV), structural variations (SVs) and extrachromosomal DNA (ecDNA) in cancer cells, revealing clonal dynamics and other oncogenic events during treatment. We further refined this technique to allow for joint profiling of chromatin architecture and transcriptome in single cells, facilitating a more comprehensive exploration of the links between chromatin architecture and gene expression in both normal tissues and tumors. Thus, Droplet Hi-C not only addresses critical gaps in chromatin analysis of heterogeneous tissues but also emerges as a versatile tool enhancing our understanding of gene regulation in health and disease.

Glioblastoma Mesenchymal Transition and Invasion are Dependent on a NF-κB/BRD2 Chromatin Complex.

Glioblastoma (GBM) represents the most aggressive subtype of glioma, noted for its profound invasiveness and molecular heterogeneity. The mesenchymal (MES) transcriptomic subtype is frequently associated with therapy resistance, rapid recurrence, and increased tumor-associated macrophages. Notably, activation of the NF-κB pathway and alterations in the PTEN gene are both associated with this malignant transition. Although PTEN aberrations have been shown to be associated with enhanced NF-κB signaling, the relationships between PTEN, NF-κB and MES transition are poorly understood in GBM. Here, we show that PTEN regulates the chromatin binding of bromodomain and extraterminal (BET) family proteins, BRD2 and BRD4, mediated by p65/RelA localization to the chromatin. By utilizing patient-derived glioblastoma stem cells and CRISPR gene editing of the RELA gene, we demonstrate a crucial role for RelA lysine 310 acetylation in recruiting BET proteins to chromatin for MES gene expression and GBM cell invasion upon PTEN loss. Remarkably, we found that BRD2 is dependent on chromatin associated acetylated RelA for its recruitment to MES gene promoters and their expression. Furthermore, loss of BRD2 results in the loss of MES signature, accompanied by an enrichment of proneural signature and enhanced therapy responsiveness. Finally, we demonstrate that disrupting the NFκB/BRD2 interaction with a brain penetrant BET-BD2 inhibitor reduces mesenchymal gene expression, GBM invasion, and therapy resistance in GBM models. This study uncovers the role of hitherto unexplored PTEN-NF-κB-BRD2 pathway in promoting MES transition and suggests inhibiting this complex with BET-BD2 specific inhibitors as a therapeutic approach to target the MES phenotype in GBM.

PTEN loss drives resistance to the neddylation inhibitor MLN4924 in glioblastoma and can be overcome with TOP2A inhibitors.

BACKGROUND: Neddylation inhibition, affecting posttranslational protein function and turnover, is a promising therapeutic approach to cancer. We report vulnerability to MLN4924 or pevonedistat (a neddylation inhibitor) in a subset of glioblastoma (GBM) preclinical models and identify biomarkers, mechanisms, and signatures of differential response. METHODS: GBM sequencing data were queried for genes associated with MLN4924 response status; candidates were validated by molecular techniques. Time-course transcriptomics and proteomics revealed processes implicated in MLN4924 response. RESULTS: Vulnerability to MLN4924 is associated with elevated S-phase populations, re-replication, and DNA damage. Transcriptomics and shotgun proteomics depict PTEN signaling, DNA replication, and chromatin instability pathways as significant differentiators between sensitive and resistant models. Loss of PTEN and its nuclear functions is associated with resistance to MLN4924. Time-course proteomics identified elevated TOP2A in resistant models through treatment. TOP2A inhibitors combined with MLN4924 prove synergistic. CONCLUSIONS: We show that PTEN status serves as both a novel biomarker for MLN4924 response in GBM and reveals a vulnerability to TOP2A inhibitors in combination with MLN4924.

Distinct pathways of homologous recombination controlled by the SWS1-SWSAP1-SPIDR complex.

Homology-directed repair (HDR), a critical DNA repair pathway in mammalian cells, is complex, leading to multiple outcomes with different impacts on genomic integrity. However, the factors that control these different outcomes are often not well understood. Here we show that SWS1-SWSAP1-SPIDR controls distinct types of HDR. Despite their requirement for stable assembly of RAD51 recombinase at DNA damage sites, these proteins are not essential for intra-chromosomal HDR, providing insight into why patients and mice with mutations are viable. However, SWS1-SWSAP1-SPIDR is critical for inter-homolog HDR, the first mitotic factor identified specifically for this function. Furthermore, SWS1-SWSAP1-SPIDR drives the high level of sister-chromatid exchange, promotes long-range loss of heterozygosity often involved with cancer initiation, and impels the poor growth of BLM helicase-deficient cells. The relevance of these genetic interactions is evident as SWSAP1 loss prolongs Blm-mutant embryo survival, suggesting a possible druggable target for the treatment of Bloom syndrome.

Are emissions of black carbon from gasoline vehicles underestimated? Insights from near and on-road measurements.

Measurements of black carbon (BC) with a high-sensitivity laser-induced incandescence (HS-LII) instrument and a single particle soot photometer (SP2) were conducted upwind, downwind, and while driving on a highway dominated by gasoline vehicles. The results are used with concurrent CO(2) measurements to derive fuel-based BC emission factors for real-world average fleet and heavy-duty diesel vehicles separately. The derived emission factors from both instruments are compared, and a low SP2 bias (relative to the HS-LII) is found to be caused by a BC mass mode diameter less than 75 nm, that is most prominent with the gasoline fleet but is not present in the heavy-duty diesel vehicle exhaust on the highway. Results from both the LII and the SP2 demonstrate that the BC emission factors from gasoline vehicles are at least a factor of 2 higher than previous North American measurements, and a factor of 9 higher than currently used emission inventories in Canada, derived with the MOBILE 6.2C model. Conversely, the measured BC emission factor for heavy-duty diesel vehicles is in reasonable agreement with previous measurements. The results suggest that greater attention must be paid to black carbon from gasoline engines to obtain a full understanding of the impact of black carbon on air quality and climate and to devise appropriate mitigation strategies.

Disparate geographic prevalences of asthma, allergic rhinoconjunctivitis and atopic eczema among adolescents in five Canadian cities.

To assess concordance of prevalence rates of asthma, allergic rhinoconjunctivitis and atopic eczema symptoms among adolescents in five Canadian cities. The International Study of Asthma and Allergies in Childhood Phase 3 written questionnaires were answered by 8334 adolescents aged 13 to 14 in Vancouver, Saskatoon, Winnipeg, Hamilton and Halifax, Canada. Prevalence rates of current symptoms ranged from 13.7-33.0% for wheezing, 14.6-22.6% for allergic rhinoconjunctivitis and 8.2-10.4% for atopic eczema. Using Hamilton as reference, the prevalence of wheezing was significantly higher in Halifax (OR = 1.58; 95% CI 1.36-1.84) and Saskatoon (1.27; 1.07-1.50) and significantly lower in Vancouver (0.51; 0.44-0.59). In contrast, allergic rhinoconjunctivitis was significantly more prevalent in Winnipeg (1.39; 1.16-1.68) and Halifax (1.36; 1.14-1.61) and trended lower in Saskatoon (0.81; 0.66-1.00). Atopic eczema was significantly more prevalent in Winnipeg (1.31; 1.01-1.69) and Vancouver (1.28; 1.04-1.58). Multivariable logistic regression analyses showed the region of residence, being born in Canada, recent use of acetaminophen and heavy exposure to traffic exhaust were significantly associated with all three allergic conditions, while obesity and having two or more smokers at home was only associated with increased risk for wheezing. Chinese ethnicity decreased that risk. Among five Canadian centres, the highest prevalence rates of allergic rhinoconjunctivitis or atopic eczema were not observed in the same regions as the highest prevalence rates of wheezing. This disparity in regional variations in the prevalence rates suggests dissimilar risk factors for the development or expression of wheezing (asthma), allergic rhinoconjunctivitis and atopic eczema.

Posterior cervical spine surgery for radiculopathy.

It is now common knowledge that cervical radiculopathy, frequently caused by disc herniation and/or degeneration, will often improve without surgical intervention. Only a small percentage of patients with the severity of symptoms necessitate surgical treatment. Surgery for radiculopathy is indicated for motor weakness, progressive neurological deficits, and progressive symptoms that do not improve with nonoperative treatment. Advantages and disadvantages exist for both ventral and dorsal approaches in the surgical treatment of cervical radiculopathy. Indications and results for dorsal nerve root decompression are discussed, and a review of our preferred techniques, including use of minimally invasive technology, is presented.

Detection of esophageal perforation using intraesophageal dye injection.

OBJECTIVES: Esophageal perforation complicating anterior cervical spine surgery is a potentially fatal complication. Early identification and immediate treatment may lower adverse effects for the patient. The purpose of this study is to assess the efficacy of intraesophageal dye injection to detect an esophageal injury and to test two novel techniques. METHOD: Ten cadaveric specimens were dissected using an anteromedial Smith-Robinson approach. Each was sequentially tested by a control and three dye injection techniques: technique A: nasogastric tube alone; technique B: nasogastric tube plus a distally placed Foley catheter; technique C: proximal plus a distally placed Foley catheter. Each technique was tested against esophageal perforations created by needle puncture (21-gauge, 18-gauge, and 14-gauge) and by a 2-mm high-speed burr. Dye visualization was independently graded as present or absent by two authors. RESULTS: In the control trial, no dye leak was visualized in any of the 10 specimens. In technique A, 0 of 10 21-gauge perforations, 1 of 10 18-gauge perforations, 2 of 10 14-gauge perforations, and 6 of 10 burr perforations were visualized. In technique B, 1 of 10 21-gauge perforations, 8 of 10 18-gauge perforations, 9 of 10 14-gauge perforations, and 9 of 10 burr perforations were visualized. In technique C, 0 of 10 21-gauge perforations, 9 of 10 18-gauge perforations, 10 of 10 14-gauge perforations, and 7 of 10 burr perforations were visualized. CONCLUSIONS: The study suggests that intraesophageal dye injection via nasogastric tube alone should not be relied upon to exclude the presence of esophageal perforation. Two novel techniques showed an improved, though limited, capability of detecting esophageal perforations.

Increased risk of postoperative neurologic deficit for spinal surgery patients with unobtainable intraoperative evoked potential data.

STUDY DESIGN: This was a retrospective study of 4,310 patients undergoing spinal surgery between 1994 and 2003. OBJECTIVES: To examine the incidence and potential causality of unobtainable somatosensory evoked potential (SSEP) and neurogenic mixed evoked potential (NMEP) data for a population of spinal surgery patients. SUMMARY OF BACKGROUND DATA: Patients with absent or unobtainable evoked potential data may increase the risk of undetected neurologic injury. To date, a comprehensive review of this patient population has not been reported. METHODS: A total of 4,310 consecutive orthopedic spinal surgeries at one institution from January 1994 through December 2003 were reviewed. Cases lacking sufficient monitoring data, despite functional neural integrity (ambulators, intact sensation), were identified. Diagnoses were divided into six general categories. The association between absent evoked potential data and associated neurologic and/or medical pathology was evaluated. RESULTS: A total of 59 of 4,310 cases (1.37%) had absent SSEP and/or NMEP intraoperative data despite functional neural integrity (44 ambulators/15 nonambulators)" 5.08% of study patients awoke with increased neurologic deficit (3 of 59), 2 global deficits, and 1 nerve root deficit. The incidence of postoperative neurologic deficit in the entire surgical population was 0.77% (33 of 4,310), 8 global (0.19%), and 25 nerve root deficits (0.058%). A Fisher's exact test demonstrated a statistically significant difference between the incidence in these two populations (P = 0.0121) and the incidence of global paraplegic deficits (P = 0.0075). CONCLUSION: Patients with unobtainable data pose a much higher risk (P = 0.0121) for postoperative neurologic deficits. Multiple Stagnara wake-up tests are strongly recommended when evoked potential data cannot be obtained.

Differences in the work-up and treatment of conditions associated with low back pain by patient gender and ethnic background.

STUDY DESIGN: Retrospective review comparing physician workup of degenerative lumbosacral pathologies between different genders and ethnic groups. OBJECTIVES: To investigate whether patient ethnicity and gender influence the workup and treatment of degenerative spinal pathologies. SUMMARY OF BACKGROUND DATA: Data from numerous studies suggest that patient gender and ethnicity play a role in medical decision-making, with white males receiving more frequent interventions than women and minorities. METHODS: Patients enrolled for an "initial visit" in the National Spine Network database with lumbosacral level degenerative diagnosis were reviewed. Variables included patient gender, ethnicity, age, duration of symptoms, patient-graded severity of symptoms, radicular symptom pattern, and work status. RESULTS: We identified 5690 patients with degenerative lumbosacral pathologies. Although females were more likely than males to have imaging tests ordered, male (18.5%) patients were significantly more likely to have surgery recommended than female (16.3%) patients (P < 0.031). Nonwhite females were 52% less likely to have surgery offered at initial visit, as compared to white males (P < 0.005). More imaging tests were ordered or reviewed among whites (76.6%) than among any other ethnic group (P = 0.162). White (18.3%) and Asian (22.5%) patients were significantly more likely to have surgery recommended or prescribed than black (11.1%) and Hispanic (14.5) patients (P < 0.0001). CONCLUSIONS: This study suggests that ethnicity and gender affect the workup and surgical management of degenerative spinal disorders. However, it should be noted that there are a number of confounding factors not identified in the database, including managed care and insurance status and cultural differences, which may affect both test ordering and treatment recommendations. Further study of bias in clinical decision-making is indicated to assure equal delivery of quality care.

Percutaneous reduction of incipient malunion of phalangeal neck fractures in children.

PURPOSE: In this article we describe our treatment of partially healed malaligned fractures of the phalangeal neck in children. METHODS: This was a retrospective study of 8 pediatric patients with phalangeal neck fractures who presented late for care to Children's Hospital in Boston. All patients were diagnosed clinically and radiographically with advanced partial healing of their fracture in a malunited position of extension. The 8 patients were followed up until fracture healing and restoration of normal motion and function were achieved. RESULTS: A K-wire was inserted into the fracture site through the dorsal callus and used to mobilize the fracture fragment by breaking down the fracture callus and partially healed bone. The wire then was used as a lever arm for fracture reduction. A separate wire was used to pin the fracture percutaneously. CONCLUSIONS: This treatment corrects the loss of flexion that occurs with a malunion in the phalangeal subcondylar fossa. It is a minimally invasive procedure that avoids the soft-tissue dissection of an open procedure.

Synthesis and cytotoxicity of substituted ethyl 2-phenacyl-3-phenylpyrrole-4-carboxylates.

The substituted ethyl-2-phenacyl-3-phenylpyrrole-4-carboxylates were synthesized by a condensation of a beta-chloroenal and an alpha-aminoketone under neutral conditions. They proved to be potent cytotoxic agents against the growth of murine L1210 and P388 leukemias and human HL-60 promyelocytic leukemia, HuT-78 lymphoma, and HeLa-S(3) uterine carcinoma. Selective compounds were active against the growth of Tmolt(3) and Tmolt(4) leukemias and THP-1 acute monocytic leukemia, liver Hepe-2, ovary 1-A9, ileum HCT-8 adenocarcinoma, and osteosarcoma HSO. A mode of action study in HL-60 cells demonstrated that DNA and protein syntheses were inhibited after 60 min at 100 microM. DNA and RNA polymerases, PRPP-amido transferase, dihydrofolate reductase, thymidylate synthase, and TMP kinase activities were interfered with by the agent with reduction of d[NTP] pools. Nonspecific interaction with the bases of DNA and cross-linking of the DNA may play a role in the mode of action of these carboxylates.