Jason Diaz

Merkel Cell Polyomavirus Large T Antigen Disrupts Host Genomic Integrity and Inhibits Cellular Proliferation

ABSTRACT Clonal integration of Merkel cell polyomavirus (MCV) DNA into the host genome has been observed in at least 80% of Merkel cell carcinoma (MCC). The integrated viral genome typically carries mutations that truncate the C-terminal DNA binding and helicase domains of the MCV large T antigen (LT), suggesting a selective pressure to remove this MCV LT region during tumor development. In this study, we show that MCV infection leads to the activation of host DNA damage responses (DDR). This activity was mapped to the C-terminal helicase-containing region of the MCV LT. The MCV LT-activated DNA damage kinases, in turn, led to enhanced p53 phosphorylation, upregulation of p53 downstream target genes, and cell cycle arrest. Compared to the N-terminal MCV LT fragment that is usually preserved in mutants isolated from MCC tumors, full-length MCV LT shows a decreased potential to support cellular proliferation, focus formation, and anchorage-independent cell growth. These apparently antitumorigenic effects can be reversed by a dominant-negative p53 inhibitor. Our results demonstrate that MCV LT-induced DDR activates p53 pathway, leading to the inhibition of cellular proliferation. This study reveals a key difference between MCV LT and simian vacuolating virus 40 LT, which activates a DDR but inhibits p53 function. This study also explains, in part, why truncation mutations that remove the MCV LT C-terminal region are necessary for the oncogenic progression of MCV-associated cancers.

Perturbation of BRD4 Protein Function by BRD4-NUT Protein Abrogates Cellular Differentiation in NUT Midline Carcinoma

NUT midline carcinoma (NMC) belongs to a class of highly lethal and poorly differentiated epithelial cancers arising mainly in human midline organs. NMC is caused by the chromosome translocation-mediated fusion of the NUT (nuclear protein in testis) gene on chromosome 15 to a few other genes, most frequently the BRD4 gene on chromosome 19. The mechanism by which the BRD4-NUT fusion product blocks NMC cellular differentiation and contributes to oncogenesis remains elusive. In this study, we show that BRD4-NUT and BRD4 colocalize in discrete nuclear foci that are hyperacetylated but transcriptionally inactive. BRD4-NUT recruits histone acetyltransferases to induce histone hyperacetylation in these chromatin foci, which provide docking sites for accumulation of additional BRD4 and associated P-TEFB (positive transcription elongation factor b) complexes in the transcriptionally inactive BRD4-NUT foci. These molecular events lead to repression of a BRD4·P-TEFB downstream target gene c-fos, a component of activator protein 1 (AP-1), that directly regulates epithelial differentiation. Knockdown of BRD4-NUT in NMC cells disperses the transcriptionally inactive chromatin foci and releases the transcriptional activators to stimulate c-fos expression, leading to restoration of cellular differentiation. Our study provides a novel mechanism by which the BRD4-NUT oncogene perturbs BRD4 functions to block cellular differentiation and to contribute to the oncogenic progression in the highly aggressive NMC.

Transoral Robotic Surgery of the Skull Base: A Cadaver and Feasibility Study

Objective: The goal of this study was to determine the potential role as well as the current limitations of the da Vinci Surgical System robot in transoral surgery of the skull base. Methods: The da Vinci robot was used to perform dissections of the skull base on 7 cadaver heads with their neck and clavicles intact. Neurosurgeons and otolaryngologists familiar with all facets of the open microscopic, minimally invasive, endoscopic and transoral robotic surgical procedure proceeded with the approach to and dissection of the human skull base. Results: The da Vinci robot provided superb illumination and 3-dimensional depth perception. The 30- degree endoscope improved cephalad visualization, and the ‘intuitive’ nature of the da Vinci surgical robot arms provided an advantage by their ability to suture the dura at the level of the clivus. An entirely transoral route provides access to the middle and lower clivus as well as the infratemporal fossa, but access to the sellar region and anterior cranial fossa is limited via a purely transoral route. Tremor-free dural closure was successfully performed. Conclusion: Our findings suggest that transoral robotic surgery utilizing the da Vinci robot system holds great potential for skull base surgical resection of extradural and intradural tumors of the middle and lower clivus and infratemporal fossa. A collaborative approach with neurosurgeon and otolaryngologist alternating at the master console and bedside is a successful strategy. Further instrument development is necessary, and continued investigation is warranted.

Phosphorylation of Merkel Cell Polyomavirus Large Tumor Antigen at Serine 816 by ATM Kinase Induces Apoptosis in Host Cells

Background: Phosphorylation regulates Merkel cell polyomavirus (MCV) large tumor antigen (LT) activity. Results: MCV LT is phosphorylated by ATM kinase at Ser-816. Conclusion: Ser-816 phosphorylation by ATM allows MCV LT to arrest cell growth and induce apoptosis. Significance: Ser-816 phosphorylation-induced apoptosis may explain why the C-terminal domain of LT is negatively selected in MCV-related tumors. Merkel cell carcinoma is a highly aggressive form of skin cancer. Merkel cell polyomavirus (MCV) infection and DNA integration into the host genome correlate with 80% of all Merkel cell carcinoma cases. Integration of the MCV genome frequently results in mutations in the large tumor antigen (LT), leading to expression of a truncated LT that retains pRB binding but with a deletion of the C-terminal domain. Studies from our laboratory and others have shown that the MCV LT C-terminal helicase domain contains growth-inhibiting properties. Additionally, we have shown that host DNA damage response factors are recruited to viral replication centers. In this study, we identified a novel MCV LT phosphorylation site at Ser-816 in the C-terminal domain. We demonstrate that activation of the ATM pathway stimulated MCV LT phosphorylation at Ser-816, whereas inhibition of ATM kinase activity prevented LT phosphorylation at this site. In vitro phosphorylation experiments confirmed that ATM kinase is responsible for phosphorylating MCV LT at Ser-816. Finally, we show that ATM kinase-mediated MCV LT Ser-816 phosphorylation may contribute to the anti-tumorigenic properties of the MCV LT C-terminal domain.

Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT’s ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells.

Brd4-mediated nuclear retention of the papillomavirus E2 protein contributes to its stabilization in host cells.

Papillomavirus E2 is a multifunctional viral protein that regulates many aspects of the viral life cycle including viral episome maintenance, transcriptional activation, and repression. E2 is degraded by the ubiquitin-proteasome pathway. Cellular bromodomain protein Brd4 has been implicated in the stabilization of the E2 protein. E2 normally shuttles between the cytoplasm and the nucleus. In this study, we demonstrate that E2 ubiquitylation mostly occurs in the cytoplasm. We also find that the interaction with Brd4 promotes nuclear retention of papillomavirus E2 proteins and contributes to their stabilization in the nucleus. Compared to wild type E2 proteins, nuclear-localization-defective mutants are rapidly degraded by the ubiquitin-proteasome pathway; however, co-expression of Brd4 redirects these mutants into the nucleus and significantly increases their stability. We further demonstrate that tethering E2 proteins to chromatin as either double-bromodomain fusion proteins or histone 2B (H2B) fusion proteins significantly stabilizes the E2 proteins. Our studies suggest that chromatin recruitment of the E2 protein via interaction with Brd4 prevents E2 ubiquitylation and proteasomal degradation in the cytoplasm, leading to its stabilization in the nucleus. These studies bring new insights for understanding Brd4-mediated E2 stabilization, and provide an additional mechanism by which the chromatin-associated Brd4 regulates E2 functions.

Full-Thickness Scalp Defects Reconstructed With Outer Table Calvarial Decortication and Surface Grafting.

Full-Thickness Scalp Defects Reconstructed With Outer Table Calvarial Decortication and Surface Grafting Reconstruction of full-thickness scalp defects is critical to avoid calvarial desiccation, sequestration, and sepsis.1 When a surgeon is faced with a scalp defect caused by a malignant tumor, several factors must be considered during a planned reconstruction, including the location of the defect, pathologic features of the tumor, margin status, and the patient’s comorbidities. However, these factors may preclude use of a large local flap or free tissue transfer, and an alternative method of reconstruction should be considered.2 Surface grafting is a quick, easy, and reliable form of reconstruction for scalp defects. However, grafts require a vascular bed to provide a nutrient blood supply. Previous authors have described the use of a single-stage approach for repair of these wounds by removing the outer table with immediate application of a split-thickness skin graft, an acellular dermis, an artificial dermis, or a wound vacuum-assisted closure.3-6 For select patients (ie, anticipated high surgical morbidity, tight scalp location, significant comorbidities, uncertain margin status, or aggressive pathologic features), reconstruction of fullthickness scalp defects can be successfully performed with outer table calvarial decortication and surface grafting.

Hexakis(acetonitrile-κN)ruthenium(II) bis­(hexa­bromo­carbadodeca­borate) aceto­nitrile solvate

The title compound, [Ru(NCCH3)6](CH6B11Br6)2·CH3CN, consists of the ’naked’ ruthenium(II) cation surrounded by six acetonitrile molecules, each coordinated via the nitrogen atoms in a linear or nearly-linear fashion in a typical octahedral over-all arrangement. The cation is balanced by the two hexa-bromocarborane cage anionic fragments [CB11H6Br6]. Weak C—H⋯Br and B—H⋯Br interactions link neighboring anions.

Reconstruction of Oromandibular Defects after Prior Irradiation Using a Temporalis Muscle and Temporoparietal Fascia Flap

Objectives: Reconstruction of oromandibular defects in the setting of previous treatment, or significant patient comorbidities, presents a significant challenge. Although free tissue transfer has shown success, it is not without considerable risk, especially in patients with poor baseline functional status. In these patients, regional pedicled flaps may provide a more suitable alternative. The combined temporalis muscle and temporoparietal fascia flap is a versatile option for oromandibular reconstruction in a previously treated field, or in patients with severe comorbidities and poor functional status. Our objective was to report our experience using a combined temporalis muscle and temporoparietal fascia flap for reconstruction of oromandibular defects in high-risk situations. Methods: Three patients were identified, medical records were reviewed, and their clinical courses were described. Functional outcomes were reviewed. We include a discussion of the relevant surgical anatomy and operative technique. All patients had previously undergone extensive treatment. They needed additional ablative surgery for different reasons: new malignancy, intractable trismus, and osteoradionecrosis. A combined temporalis muscle and temporoparietal fascia flap was used to reconstruct the oromandibular defects in each patient. Results: All flaps survived. Functional status improved in all patients. There were no significant operative or postoperative complications. Conclusions: The combined temporalis muscle and temporoparietal fascia flap provides a reliable option for reconstruction of complex oromandibular defects in high-risk situations. In previously treated fields, the transfer of a vascularized flap into the wound hastens healing and improves the quality of native oral cavity tissue.