MYC Controls the Epstein-Barr Virus Lytic Switch.

Epstein-Barr virus (EBV) is associated with multiple human malignancies. To evade immune detection, EBV switches between latent and lytic programs. How viral latency is maintained in tumors or in memory B cells, the reservoir for lifelong EBV infection, remains incompletely understood. To gain insights, we performed a human genome-wide CRISPR/Cas9 screen in Burkitt lymphoma B cells. Our analyses identified a network of host factors that repress lytic reactivation, centered on the transcription factor MYC, including cohesins, FACT, STAGA, and Mediator. Depletion of MYC or factors important for MYC expression reactivated the lytic cycle, including in Burkitt xenografts. MYC bound the EBV genome origin of lytic replication and suppressed its looping to the lytic cycle initiator BZLF1 promoter. Notably, MYC abundance decreases with plasma cell differentiation, a key lytic reactivation trigger. Our results suggest that EBV senses MYC abundance as a readout of B cell state and highlights Burkitt latency reversal therapeutic targets.

Long-read sequencing reveals complex patterns of wraparound transcription in polyomaviruses.

Polyomaviruses (PyV) are ubiquitous pathogens that can cause devastating human diseases. Due to the small size of their genomes, PyV utilize complex patterns of RNA splicing to maximize their coding capacity. Despite the importance of PyV to human disease, their transcriptome architecture is poorly characterized. Here, we compare short- and long-read RNA sequencing data from eight human and non-human PyV. We provide a detailed transcriptome atlas for BK polyomavirus (BKPyV), an important human pathogen, and the prototype PyV, simian virus 40 (SV40). We identify pervasive wraparound transcription in PyV, wherein transcription runs through the polyA site and circles the genome multiple times. Comparative analyses identify novel, conserved transcripts that increase PyV coding capacity. One of these conserved transcripts encodes superT, a T antigen containing two RB-binding LxCxE motifs. We find that superT-encoding transcripts are abundant in PyV-associated human cancers. Together, we show that comparative transcriptomic approaches can greatly expand known transcript and coding capacity in one of the simplest and most well-studied viral families.

Restriction of Replication of Oncolytic Herpes Simplex Virus with a Deletion of γ34.5 in Glioblastoma Stem-Like Cells.

Oncolytic viruses, including herpes simplex viruses (HSVs), are a new class of cancer therapeutic engineered to infect and kill cancer cells while sparing normal tissue. To ensure that oncolytic HSV (oHSV) is safe in the brain, all oHSVs in clinical trial for glioma lack the γ34.5 genes responsible for neurovirulence. However, loss of γ34.5 attenuates growth in cancer cells. Glioblastoma (GBM) is a lethal brain tumor that is heterogeneous and contains a subpopulation of cancer stem cells, termed GBM stem-like cells (GSCs), that likely promote tumor progression and recurrence. GSCs and matched serum-cultured GBM cells (ScGCs), representative of bulk or differentiated tumor cells, were isolated from the same patient tumor specimens. ScGCs are permissive to replication and cell killing by oHSV with deletion of the γ34.5 genes (γ34.5- oHSV), while patient-matched GSCs were not, implying an underlying biological difference between stem and bulk cancer cells. GSCs specifically restrict the synthesis of HSV-1 true late (TL) proteins, without affecting viral DNA replication or transcription of TL genes. A global shutoff of cellular protein synthesis also occurs late after γ34.5- oHSV infection of GSCs but does not affect the synthesis of early and leaky late viral proteins. Levels of phosphorylated eIF2α and eIF4E do not correlate with cell permissivity. Expression of Us11 in GSCs rescues replication of γ34.5- oHSV. The difference in degrees of permissivity between GSCs and ScGCs to γ34.5- oHSV illustrates a selective translational regulatory pathway in GSCs that may be operative in other stem-like cells and has implications for creating oHSVs.IMPORTANCE Herpes simplex virus (HSV) can be genetically engineered to endow cancer-selective replication and oncolytic activity. γ34.5, a key neurovirulence gene, has been deleted in all oncolytic HSVs in clinical trial for glioma. Glioblastoma stem-like cells (GSCs) are a subpopulation of tumor cells thought to drive tumor heterogeneity and therapeutic resistance. GSCs are nonpermissive for γ34.5- HSV, while non-stem-like cancer cells from the same patient tumors are permissive. GSCs restrict true late protein synthesis, despite normal viral DNA replication and transcription of all kinetic classes. This is specific for true late translation as early and leaky late transcripts are translated late in infection, notwithstanding shutoff of cellular protein synthesis. Expression of Us11 in GSCs rescues the replication of γ34.5- HSV. We have identified a cell type-specific innate response to HSV-1 that limits oncolytic activity in glioblastoma.

Monolithically integrated L-band PICs and transceiver modules with 6λ x 200 Gbps (1.2 Tbps) for C + L band communication systems.

We present monolithically integrated multi-channel coherent L-band transmitter (Tx) and receiver (Rx) photonic integrated circuits (PICs) on InP substrates. The L-band PICs are able to provide post-forward error correction (FEC), error-free operation for dual-polarization (DP) 16-QAM coherent transmission at 33 Gbaud. These transceivers operate at 200 Gbps per channel and support 1.2 Tbps aggregate capacity per 6 channel PIC. We also demonstrate in this work a C + L band communication system with two C-band superchannels (2 x 6λ) and three L-band superchannels (3 x 6λ) over a 600 km link. The received signals all have Q > 7.7 dB, which is well above the error-free threshold of the FEC used in this work.

Room-Temperature Spin Polariton Diode Laser.

A spin-polarized laser offers inherent control of the output circular polarization. We have investigated the output polarization characteristics of a bulk GaN-based microcavity polariton diode laser at room temperature with electrical injection of spin-polarized electrons via a FeCo/MgO spin injector. Polariton laser operation with a spin-polarized current is characterized by a threshold of ∼69 A/cm^{2} in the light-current characteristics, a significant reduction of the electroluminescence linewidth and blueshift of the emission peak. A degree of output circular polarization of ∼25% is recorded under remanent magnetization. A second threshold, due to conventional photon lasing, is observed at an injection of ∼7.2 kA/cm^{2}. The variation of output circular and linear polarization with spin-polarized injection current has been analyzed with the carrier and exciton rate equations and the Gross-Pitaevskii equations for the condensate and there is good agreement between measured and calculated data.

Formation and nature of InGaN quantum dots in GaN nanowires.

InGaN/GaN disk-in-nanowire heterostructures on silicon substrates have emerged as important gain media for the realization of visible light sources. The nature of quantum confinement in the disks is largely unknown. From the unique nature of the measured temperature dependence of the radiative lifetime and direct transmission electron microscopy, it is evident that such self-organized islands (disks) behave as quantum dots. This is confirmed by the observation of single photon emission from a single disk-in-nanowire and the presence of a sharp minimum in the line width enhancement factor of edge emitting lasers having the InGaN disks as the gain media.

Physical model for high indium content InGaN/GaN self-assembled quantum dot ridge-waveguide lasers emitting at red wavelengths (λ ~ 630 nm).

We present a physical model for recently demonstrated high indium content self-assembled In0.4Ga0.6N/GaN quantum dot (QD)-based ridge-waveguide lasers emitting at red wavelengths. The strain distribution in the QD is calculated using linear elastic theory with the application of shrink-fit boundary condition at the InGaN/GaN material interface, and the electronic states are evaluated using a single-band effective mass Hamiltonian. A Schrödinger-Poisson self-consistent solver is used to describe the effect of charge screening under current injection. Our theoretical result shows a good match to the measured Hakki-Paoli gain spectrum. Combining the calculated gain spectrum and cavity properties, we have developed a device-level simulation to successfully explain the electrical and optical characteristics of this specific laser. Possible solutions to improving the device performance have been explored.

Optical constants of In(x)Ga(1-x)N (0 ≤ x ≤ 0.73) in the visible and near-infrared wavelength regimes.

Complex refractive indices of In(x)Ga(1-x)N epitaxial layers have been determined from analysis of data obtained by spectroscopic ellipsometry. The measurements were made in the wavelength range of 400-1687 nm. The samples were grown by plasma-assisted molecular beam epitaxy on (001) silicon substrate and are of the wurtzite crystalline form. A comparison of the fundamental absorption edge derived from analysis of measured data and the measured photoluminescence peak emission energy indicates a Stokes shift present in the alloys.

Monolithic electrically injected nanowire array edge-emitting laser on (001) silicon.

A silicon-based laser, preferably electrically pumped, has long been a scientific and engineering goal. We demonstrate here, for the first time, an edge-emitting InGaN/GaN disk-in-nanowire array electrically pumped laser emitting in the green (λ = 533 nm) on (001) silicon substrate. The devices display excellent dc and dynamic characteristics with values of threshold current density, differential gain, T0 and small signal modulation bandwidth equal to 1.76 kA/cm(2), 3 × 10(-17) cm(2), 232 K, and 5.8 GHz respectively under continuous wave operation. Preliminary reliability measurements indicate a lifetime of 7000 h. The emission wavelength can be tuned by varying the alloy composition in the quantum disks. The monolithic nanowire laser on (001)Si can therefore address wide-ranging applications such as solid state lighting, displays, plastic fiber communication, medical diagnostics, and silicon photonics.

Detailed model for the In0.18Ga0.82N/GaN self-assembled quantum dot active material for λ = 420 nm emission.

We present a comprehensive model for In(0.18)Ga(0.82)N/GaN self-assembled quantum dot (QD) active material. The strain distribution in the QD structure is studied using linear elastic theory with the application of the shrink-fit boundary condition at the material interface. Subsequent calculations also predict the strain-induced quantum-confined Stark effect (QCSE). Under carrier injection, the overall effect of band bending and charge screening is studied by solving the Schrödinger and Poisson equations self-consistently. The optical gain spectrum of the InGaN/GaN QD active material is calculated based on the electronic states solved from the Schrödinger-Poisson equation, and both the calculated material gain peak and emission wavelength agree well with the measured experimental data.

Room temperature electrically injected polariton laser.

Room temperature electrically pumped inversionless polariton lasing is observed from a bulk GaN-based microcavity diode. The low nonlinear threshold for polariton lasing occurs at 169 A/cm(2) in the light-current characteristics, accompanied by a collapse of the emission linewidth and small blueshift of the emission peak. Measurement of angle-resolved luminescence, polariton condensation and occupation in momentum space, and output spatial coherence and polarization have also been made. A second threshold, due to conventional photon lasing, is observed at an injection of 44 kA/cm(2).

Enhancement of HSV-1 cell-free virion release by the envelope protein gC.

Glycoprotein C (gC), one of ∼12 HSV-1 envelope glycoproteins, carries out several important functions during infection, including the enhancement of virion attachment by binding to host cell heparan sulfate proteoglycans (HSPG). Here we report that gC can also enhance the release of cell-free progeny virions at the end of the infectious cycle. This activity was observed in multiple cellular contexts including Vero cells and immortalized human keratinocytes. In the absence of gC, progeny virions bound more tightly to infected cells, suggesting that gC promotes the detachment of virions from the infected cell surface. Given this finding, we analyzed the biochemical interactions that tether progeny virions to cells and report evidence for two distinct modes of binding. One is consistent with a direct interaction between gC and HSPG, whereas the other is gC-independent and likely does not involve HSPG. Together, our results i) identify a novel function for a long-studied HSV-1 glycoprotein, and ii) demonstrate that the extracellular release of HSV-1 virions is a dynamic process involving multiple viral and host components.

Quantum dot rolled-up microtube optoelectronic integrated circuit.

A rolled-up microtube optoelectronic integrated circuit operating as a phototransceiver is demonstrated. The microtube is made of a InGaAs/GaAs strained bilayer with InAs self-organized quantum dots inserted in the GaAs layer. The phototransceiver consists of an optically pumped microtube laser and a microtube photoconductive detector connected by an a-Si/SiO2 waveguide. The loss in the waveguide and responsivity of the entire phototransceiver circuit are 7.96 dB/cm and 34 mA/W, respectively.

YAP1 and WWTR1 expression inversely correlates with neuroendocrine markers in Merkel cell carcinoma.

BackgroundMerkel cell carcinoma (MCC) is an aggressive neuroendocrine (NE) skin cancer caused by severe UV-induced mutations or expression of Merkel cell polyomavirus (MCPyV) large and small T antigens (LT and ST). Despite deep genetic differences between MCPyV-positive and -negative subtypes, current clinical diagnostic markers are indistinguishable, and the expression profile of MCC tumors is, to our knowledge, unexplored.MethodsHere, we leveraged bulk and single-cell RNA-Seq of patient-derived tumor biopsies and cell lines to explore the underlying transcriptional environment of MCC.ResultsStrikingly, MCC samples could be separated into transcriptional subtypes that were independent of MCPyV status. Instead, we observed an inverse correlation between a NE gene signature and the Hippo pathway transcription factors Yes1-associated transcriptional regulator (YAP1) and WW domain-containing transcriptional regulator 1 (WWTR1). This inverse correlation was broadly present at the transcript and protein levels in the tumor biopsies as well as in established and patient-derived cell lines. Mechanistically, expression of YAP1 or WWTR1 in a MCPyV-positive MCC cell line induced cell-cycle arrest at least in part through TEA domain-dependent (TEAD-dependent) transcriptional repression of MCPyV LT.ConclusionThese findings identify what we believe to be a previously unrecognized heterogeneity in NE gene expression within MCC and support a model of YAP1/WWTR1 silencing as essential for the development of MCPyV-positive MCC.FundingUS Public Health Service grants R35CA232128, P01CA203655, and P30CA06516.

Merkel Cell Carcinoma Sensitivity to EZH2 Inhibition Is Mediated by SIX1 Derepression.

Polycomb repressive complex 2 has a critical role in the maintenance of bivalent promoters and is often perturbed in cancer, including neuroendocrine tumors. In this study, we investigated the susceptibility of Merkel cell carcinoma (MCC), a neuroendocrine carcinoma of the skin, to inhibitors of the Polycomb repressive complex 2 catalytic subunit EZH2. We show that a subset of MCC cell lines is sensitive to EZH2 inhibitor-induced cell viability loss. We find that inhibitor treatment of susceptible cells derepresses the Polycomb repressive complex 2 target SIX1, a transcription factor in the PAX-SIX-EYA-DACH network normally involved in inner ear hair cell development, and that PAX-SIX-EYA-DACH network transcription factors are critical contributors to EZH2 inhibitor-induced MCC cell viability loss. Furthermore, we show the EZH2 inhibitor tazemetostat slows the growth of MCC xenografts and derepresses SIX1 and its downstream inner ear transcriptional target MYO6 in vivo. We propose that EZH2 inhibition in MCC leads to SIX1 derepression with dysregulation of hearing-related transcriptional programs and growth inhibition. This study provides evidence that MCC tumors may be specifically susceptible to EZH2 inhibitors, while giving mechanistic insight into the transcriptional programs these inhibitors perturb in MCC, and potentially in other neuroendocrine cancers.

Addiction of Merkel cell carcinoma to MUC1-C identifies a potential new target for treatment.

Merkel cell carcinoma (MCC) is an aggressive malignancy with neuroendocrine (NE) features, limited treatment options, and a lack of druggable targets. There is no reported involvement of the MUC1-C oncogenic protein in MCC progression. We show here that MUC1-C is broadly expressed in MCCs and at higher levels in Merkel cell polyomavirus (MCPyV)-positive (MCCP) relative to MCPyV-negative (MCCN) tumors. Our results further demonstrate that MUC1-C is expressed in MCCP, as well as MCCN, cell lines and regulates common sets of signaling pathways related to RNA synthesis, processing, and transport in both subtypes. Mechanistically, MUC1-C (i) interacts with MYCL, which drives MCC progression, (ii) is necessary for expression of the OCT4, SOX2, KLF4, MYC, and NANOG pluripotency factors, and (iii) induces the NEUROD1, BRN2 and ATOH1 NE lineage dictating transcription factors. We show that MUC1-C is also necessary for MCCP and MCCN cell survival by suppressing DNA replication stress, the p53 pathway, and apoptosis. In concert with these results, targeting MUC1-C genetically and pharmacologically inhibits MCC self-renewal capacity and tumorigenicity. These findings demonstrate that MCCP and MCCN cells are addicted to MUC1-C and identify MUC1-C as a potential target for MCC treatment.

Milademetan is a highly potent MDM2 inhibitor in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine carcinoma of the skin with 2 etiologies. Merkel cell polyomavirus (MCPyV) integration is present in about 80% of all MCC. Virus-positive MCC (MCCP) tumors have few somatic mutations and usually express WT p53 (TP53). By contrast, virus-negative MCC (MCCN) tumors present with a high tumor mutational burden and predominantly UV mutational signature. MCCN tumors typically contain mutated TP53. MCCP tumors express 2 viral proteins: MCPyV small T antigen and a truncated form of large T antigen. MCPyV ST specifically activates expression of MDM2, an E3 ubiquitin ligase of p53, to inhibit p53-mediated tumor suppression. In this study, we assessed the efficacy of milademetan, a potent, selective, and orally available MDM2 inhibitor in several MCC models. Milademetan reduced cell viability of WT p53 MCC cell lines and triggered a rapid and sustained p53 response. Milademetan showed a dose-dependent inhibition of tumor growth in MKL-1 xenograft and patient-derived xenograft models. Here, along with preclinical data for the efficacy of milademetan in WT p53 MCC tumors, we report several in vitro and in vivo models useful for future MCC studies.

Reversal of viral and epigenetic HLA class I repression in Merkel cell carcinoma.

Cancers avoid immune surveillance through an array of mechanisms, including perturbation of HLA class I antigen presentation. Merkel cell carcinoma (MCC) is an aggressive, HLA-I-low, neuroendocrine carcinoma of the skin often caused by the Merkel cell polyomavirus (MCPyV). Through the characterization of 11 newly generated MCC patient-derived cell lines, we identified transcriptional suppression of several class I antigen presentation genes. To systematically identify regulators of HLA-I loss in MCC, we performed parallel, genome-scale, gain- and loss-of-function screens in a patient-derived MCPyV-positive cell line and identified MYCL and the non-canonical Polycomb repressive complex 1.1 (PRC1.1) as HLA-I repressors. We observed physical interaction of MYCL with the MCPyV small T viral antigen, supporting a mechanism of virally mediated HLA-I suppression. We further identify the PRC1.1 component USP7 as a pharmacologic target to restore HLA-I expression in MCC.

Epigenetic crossroads of the Epstein-Barr virus B-cell relationship.

Epstein-Barr virus (EBV) is a gamma-herpesvirus that establishes lifelong infection in the majority of people worldwide. EBV uses epigenetic reprogramming to switch between multiple latency states in order to colonize the memory B-cell compartment and to then periodically undergo lytic reactivation upon plasma cell differentiation. This review focuses on recent advances in the understanding of epigenetic mechanisms that EBV uses to control its lifecycle and to subvert the growth and survival pathways that underly EBV-driven B-cell differentiation versus B-cell growth transformation, a hallmark of the first human tumor virus. These include the formation of viral super enhancers that drive expression of key host dependency factors, evasion of tumor suppressor responses, prevention of plasmablast differentiation, and regulation of the B-cell lytic switch.