The oncogenic BRD4-NUT chromatin regulator drives aberrant transcription within large topological domains.

NUT midline carcinoma (NMC), a subtype of squamous cell cancer, is one of the most aggressive human solid malignancies known. NMC is driven by the creation of a translocation oncoprotein, BRD4-NUT, which blocks differentiation and drives growth of NMC cells. BRD4-NUT forms distinctive nuclear foci in patient tumors, which we found correlate with ∼100 unprecedented, hyperacetylated expanses of chromatin that reach up to 2 Mb in size. These "megadomains" appear to be the result of aberrant, feed-forward loops of acetylation and binding of acetylated histones that drive transcription of underlying DNA in NMC patient cells and naïve cells induced to express BRD4-NUT. Megadomain locations are typically cell lineage-specific; however, the cMYC and TP63 regions are targeted in all NMCs tested and play functional roles in tumor growth. Megadomains appear to originate from select pre-existing enhancers that progressively broaden but are ultimately delimited by topologically associating domain (TAD) boundaries. Therefore, our findings establish a basis for understanding the powerful role played by large-scale chromatin organization in normal and aberrant lineage-specific gene transcription.

Ectopic protein interactions within BRD4-chromatin complexes drive oncogenic megadomain formation in NUT midline carcinoma.

To investigate the mechanism that drives dramatic mistargeting of active chromatin in NUT midline carcinoma (NMC), we have identified protein interactions unique to the BRD4-NUT fusion oncoprotein compared with wild-type BRD4. Using cross-linking, affinity purification, and mass spectrometry, we identified the EP300 acetyltransferase as uniquely associated with BRD4 through the NUT fusion in both NMC and non-NMC cell types. We also discovered ZNF532 associated with BRD4-NUT in NMC patient cells but not detectable in 293T cells. EP300 and ZNF532 are both implicated in feed-forward regulatory loops leading to propagation of the oncogenic chromatin complex in BRD4-NUT patient cells. Adding key functional significance to our biochemical findings, we independently discovered a ZNF532-NUT translocation fusion in a newly diagnosed NMC patient. ChIP sequencing of the major players NUT, ZNF532, BRD4, EP300, and H3K27ac revealed the formation of ZNF532-NUT-associated hyperacetylated megadomains, distinctly localized but otherwise analogous to those found in BRD4-NUT patient cells. Our results support a model in which NMC is dependent on ectopic NUT-mediated interactions between EP300 and components of BRD4 regulatory complexes, leading to a cascade of misregulation.

Intensive treatment and survival outcomes in NUT midline carcinoma of the head and neck.

BACKGROUND: NUT midline carcinoma is a rare and aggressive genetically characterized subtype of squamous cell carcinoma frequently arising from the head and neck. The characteristics and optimal management of head and neck NUT midline carcinoma (HNNMC) are unclear. METHODS: A retrospective review of all known cases of HNNMC in the International NUT Midline Carcinoma Registry as of December 31, 2014, was performed. Forty-eight consecutive patients were treated from 1993 to 2014, and clinicopathologic variables and outcomes for 40 patients were available for analyses; they composed the largest HNNMC cohort studied to date. Overall survival (OS) and progression-free survival (PFS) according to patient characteristics and treatment were analyzed. RESULTS: This study identified a 5-fold increase in the diagnosis of HNNMC from 2011 to 2014. The median age was 21.9 years (range, 0.1-81.7 years); the male and female proportions were 40% and 60%, respectively; and 86% had bromodomain containing 4-nuclear protein in testis (BRD4-NUT) fusion. The initial treatment was initial surgery with or without adjuvant chemoradiation or adjuvant radiation (56%), initial radiation with or without chemotherapy (15%), or initial chemotherapy with or without surgery or radiation (28%). The median PFS was 6.6 months (range, 4.7-8.4 months). The median OS was 9.7 months (range, 6.6-15.6 months). The 2-year PFS rate was 26% (95% confidence interval [CI], 13%-40%). The 2-year OS rate was 30% (95% CI, 16%-46%). Initial surgery with or without postoperative chemoradiation or radiation (P = .04) and complete resection with negative margins (P = .01) were significant predictors of improved OS even after adjustments for age, tumor size, and neck lymphadenopathy. Initial radiation or chemotherapy and the NUT translocation type were not associated with outcomes. CONCLUSIONS: HNNMC portends a poor prognosis. Aggressive initial surgical resection with or without postoperative chemoradiation or radiation is associated with significantly enhanced survival. Chemotherapy or radiation alone is often inadequate. Cancer 2016;122:3632-40. © 2016 American Cancer Society.

High-throughput continuous-flow microfluidic electroporation of mRNA into primary human T cells for applications in cellular therapy manufacturing.

Implementation of gene editing technologies such as CRISPR/Cas9 in the manufacture of novel cell-based therapeutics has the potential to enable highly-targeted, stable, and persistent genome modifications without the use of viral vectors. Electroporation has emerged as a preferred method for delivering gene-editing machinery to target cells, but a major challenge remaining is that most commercial electroporation machines are built for research and process development rather than for large-scale, automated cellular therapy manufacturing. Here we present a microfluidic continuous-flow electrotransfection device designed for precise, consistent, and high-throughput genetic modification of target cells in cellular therapy manufacturing applications. We optimized our device for delivery of mRNA into primary human T cells and demonstrated up to 95% transfection efficiency with minimum impact on cell viability and expansion potential. We additionally demonstrated processing of samples comprising up to 500 million T cells at a rate of 20 million cells/min. We anticipate that our device will help to streamline the production of autologous therapies requiring on the order of 10[Formula: see text]-10[Formula: see text] cells, and that it is well-suited to scale for production of trillions of cells to support emerging allogeneic therapies.

Acoustophoretic rapid media exchange and continuous-flow electrotransfection of primary human T cells for applications in automated cellular therapy manufacturing.

Autologous cellular therapies based on modifying T cells to express chimeric antigen receptor genes have been highly successful in treating hematological cancers. Deployment of these therapies is limited by the complexity and costs associated with their manufacturing. Transitioning these processes from virus-based methods for gene delivery to a non-viral method, such as electroporation, has the potential to greatly reduce cost and manufacturing time while increasing safety and efficacy. Major challenges with electroporation are the negative impacts on cell health associated with exposure to high-magnitude electric fields, and that most commercial bulk electroporators are low-precision instruments designed for manually-operated, lower-throughput batch processing of cells. Negative effects on cell health can be mitigated by use of specialized electroporation medias, but this adds processing steps, and long-term exposure to these medias can reduce transfection efficiency and post-transfection viability. To enable automated, clinical-scale production of cellular therapies using electrotransfection in specialized medias, we developed a high-precision microfluidic platform that automatically and continuously transfers cells from culture media into electroporation media using acoustophoresis, and then immediately applies electric fields from integrated electrodes. This limits cell residence time in electroporation media to seconds, and enables high transfection efficiency with minimum impact on cell viability. We tested our system by transferring primary human T cells from a standard cell media to electroporation media, and then transfecting them with mRNA encoding an mCherry fluorescent protein. We achieved a media exchange efficiency of 86% and transfection efficiency of up to 60%, with less than a 5% reduction in viability.

A Microfluidic Device to Enhance Viral Transduction Efficiency During Manufacture of Engineered Cellular Therapies.

The development and approval of engineered cellular therapies are revolutionizing approaches to treatment of diseases. However, these life-saving therapies require extensive use of inefficient bioprocessing equipment and specialized reagents that can drive up the price of treatment. Integration of new genetic material into the target cells, such as viral transduction, is one of the most costly and labor-intensive steps in the production of cellular therapies. Approaches to reducing the costs associated with gene delivery have been developed using microfluidic devices to increase overall efficiency. However, these microfluidic approaches either require large quantities of virus or pre-concentration of cells with high-titer viral particles. Here, we describe the development of a microfluidic transduction device (MTD) that combines microfluidic spatial confinement with advective flow through a membrane to efficiently colocalize target cells and virus particles. We demonstrate that the MTD can improve the efficiency of lentiviral transduction for both T-cell and hematopoietic stem-cell (HSC) targets by greater than two fold relative to static controls. Furthermore, transduction saturation in the MTD is reached with only half the virus required to reach saturation under static conditions. Moreover, we show that MTD transduction does not adversely affect cell viability or expansion potential.

Micropatterned cell sheets as structural building blocks for biomimetic vascular patches.

To successfully develop a functional tissue-engineered vascular patch, recapitulating the hierarchical structure of vessel is critical to mimic mechanical properties. Here, we use a cell sheet engineering strategy with micropatterning technique to control structural organization of bovine aortic vascular smooth muscle cell (VSMC) sheets. Actin filament staining and image analysis showed clear cellular alignment of VSMC sheets cultured on patterned substrates. Viability of harvested VSMC sheets was confirmed by Live/Dead® cell viability assay after 24 and 48 h of transfer. VSMC sheets stacked to generate bilayer VSMC patches exhibited strong inter-layer bonding as shown by lap shear test. Uniaxial tensile testing of monolayer VSMC sheets and bilayer VSMC patches displayed nonlinear, anisotropic stress-stretch response similar to the biomechanical characteristic of a native arterial wall. Collagen content and structure were characterized to determine the effects of patterning and stacking on extracellular matrix of VSMC sheets. Using finite-element modeling to simulate uniaxial tensile testing of bilayer VSMC patches, we found the stress-stretch response of bilayer patterned VSMC patches under uniaxial tension to be predicted using an anisotropic hyperelastic constitutive model. Thus, our cell sheet harvesting system combined with biomechanical modeling is a promising approach to generate building blocks for tissue-engineered vascular patches with structure and mechanical behavior mimicking native tissue.