Merkel Cell Polyomavirus Small T Antigen Promotes Pro-Glycolytic Metabolic Perturbations Required for Transformation

An accurate analytic model describing the microscopic mechanism of high-harmonic generation (HHG) in solids is derived. Extensive first-principles simulations within a time-dependent density-functional framework corroborate the conclusions of the model. Our results reveal that (i) the emitted HHG spectra are highly anisotropic and laser-polarization dependent even for cubic crystals; (ii) the harmonic emission is enhanced by the inhomogeneity of the electron-nuclei potential; the yield is increased for heavier atoms; and (iii) the cutoff photon energy is driver-wavelength independent. Moreover, we show that it is possible to predict the laser polarization for optimal HHG in bulk crystals solely from the knowledge of their electronic band structure. Our results pave the way to better control and optimize HHG in solids by engineering their band structure

Dual Inhibition of MDM2 and MDM4 in Virus-Positive Merkel Cell Carcinoma Enhances the p53 Response

Merkel cell polyomavirus (MCV) contributes to approximately 80% of all Merkel cell carcinomas (MCC), a highly aggressive neuroendocrine carcinoma of the skin. MCV-positive MCC expresses small T antigen (ST) and a truncated form of large T antigen (LT) and usually contains wild type p53 (TP53) and RB (RB1). In contrast, virus-negative MCC contains inactivating mutations in TP53 and RB1. While the MCV truncated LT can bind and inhibit RB, it does not bind p53. We report here that MCV LT binds to RB leading to increased levels of ARF, an inhibitor of MDM2, and activation of p53. However, co-expression of ST reduced p53 activation. MCV ST recruits the MYC homologue MYCL (L-Myc) to the EP400 chromatin remodeler complex and transactivates specific target genes. We observed that depletion of EP400 in MCV-positive MCC cell lines led to increased p53 target gene expression. We suspected that the MCV ST-MYCL-EP400 complex could functionally inactivate p53 but the underlying mechanism was not known. Integrated ChIP and RNA-seq analysis following EP400 depletion identified MDM2 as well as CK1�, an activator of MDM4, as target genes of the ST-MYCL-EP400 complex. In addition, MCV-positive MCC cells expressed high levels of MDM4. Combining MDM2 inhibitors with lenalidomide targeting CK1� or an MDM4 inhibitor caused synergistic activation of p53 leading to an apoptotic response in MCV-positive MCC cells and MCC-derived xenografts in mice. These results support dual targeting of MDM2 and MDM4 in virus-positive MCC and other p53 wild type tumors

MCC cell lines exhibit variable ECAR and sensitivity to MCT1 inhibition.

<p><b>A)</b> ECAR (mpH/min) of MKL-1, MKL-2 and WaGa lines (minutes). Cells were treated with oligomycin (1 μM) at the indicated time point (arrow). <b>B-D)</b> XTT proliferation assay of MKL-1, MKL-2 and WaGa cells treated with either DMSO, CHC (5 mM), SR13800 (100 nM), or SR13801 (100 nM) (days).</p

MCPyV ST increases aerobic glycolysis and MCT1 sensitivity.

<p><b>A)</b> Media glucose (Glc) and lactate (Lac) levels (mM) from cultures of IMR90 cells expressing ST or GFP were measured at the indicated day following dox addition. **P < 0.005 calculated using unpaired student’s T test between the marked GFP and ST points. <b>B)</b> ECAR (mpH/min) of IMR90 cells inducibly expressing ST with and without dox addition for 48 hours. ***P < 0.0005 calculated using unpaired student’s T test. <b>C)</b> ECAR of IMR90 cells expressing ST or GFP with CHC (5 mM) or DMSO (minutes) following 48 hours of dox treatment. Cells were treated with oligomycin (1 μM) at the indicated time point. ***P < 0.0005 calculated using unpaired student’s T test between GFP-DMSO and ST-DMSO samples. <b>D)</b> OCR (pmoles/min) of cells (minutes) as in C. <b>E)</b> Growth of IMR90 cells expressing ST or GFP treated with dox and CHC or DMSO was assessed by crystal violet every day for 5 days. ***P < 0.0005 calculated using unpaired, two-tailed student’s T test between ST-DMSO and ST-CHC treatments. Key same as in C.</p

Temporal transcriptome of IMR90 fibroblasts inducibly expressing MCPyV ST.

<p><b>A)</b> IMR90 fibroblasts containing dox-inducible MCPyV ST or GFP vectors were treated with dox and harvested every 8 hours for RNA extraction. Each time point represents three biological replicas. <b>B)</b> Mean ST transcript levels and <b>C)</b> immunoblotting for ST, GFP and vinculin from cells collected every 8 hours for 96 hours following dox treatment. <b>D)</b> Hierarchical clustering and fold change between MCPyV ST and GFP following dox induction for 96 hours. Each bar represents an average of three experiments for each time point. The enrichment of “Cancer Hallmark” gene sets are represented relative to the ST-differentially expressed clusters, including epithelial to mesenchymal transition (EMT), tumor necrosis factor-α (TNFA signaling via NF-κB), hypoxia, mTORC1, oxidative phosphorylation, glycolysis, MYC, and several cell cycle clusters including E2F targets, G2M checkpoint and mitotic spindle. The color bar indicates statistical significance, yellow p < 0.05 and gray p > 0.05.</p

MYC isoforms differentially regulate glycolysis gene expression and ECAR of MCC cells.

<p><b>A)</b> MKL-1 and WaGa cells containing inducible vectors for MYC, MYCN or MYCL were treated with (+) or without (-) dox for 72 hours and lysates were immunoblotted with the indicated antibodies. <b>B)</b> ECAR (mpH/min) of MKL-1 cells inducibly expressing GFP, MYC, MYCN or MYCL after 72 hours of dox addition (minutes). Cells were treated with oligomycin (1 μM) at the indicated time point. *P < 0.05 calculated using unpaired student’s T test between MYC and MYCL samples.</p

MCPyV-transformed cells exhibit elevated ECAR and sensitivity to MCT1 inhibitors.

<p><b>A)</b> Anchorage-independent growth of IMR90 PH, PHL, PHE and PHEL cells. ****P < 0.0001 calculated using ordinary one-way ANOVA with multiple comparisons. <b>B)</b> Basal ECAR (mpH/min) measurement of p53DD, PH, PHE and PHE + MYCL (PHEL) cells. **P < 0.005 and ****P < 0.0001 calculated using ordinary one-way ANOVA with multiple comparisons. <b>C)</b> Proliferation of PHEL cells treated with DMSO, CHC (5 mM), SR13800 (100 nM), or SR13801 (100 nM) was assessed by crystal violet staining. *P < 0.05 calculated using student’s T test between DMSO-SR13800 and DMSO-SR13801 samples. <b>D)</b> Anchorage-independent growth of IMR90 PHE and PHEL cells treated with DMSO, CHC, SR13800 (SR800) or SR13801 (SR801). ****P < 0.0001 calculated using ordinary one-way ANOVA with multiple comparisons.</p

Merkel cell polyomavirus recruits MYCL to the EP400 complex to promote oncogenesis

Merkel cell carcinoma (MCC) frequently contains integrated copies of Merkel cell polyomavirus DNA that express a truncated form of Large T antigen (LT) and an intact Small T antigen (ST). While LT binds RB and inactivates its tumor suppressor function, it is less clear how ST contributes to MCC tumorigenesis. Here we show that ST binds specifically to the MYC homolog MYCL (L-MYC) and recruits it to the 15-component EP400 histone acetyltransferase and chromatin remodeling complex. We performed a large-scale immunoprecipitation for ST and identified co-precipitating proteins by mass spectrometry. In addition to protein phosphatase 2A (PP2A) subunits, we identified MYCL and its heterodimeric partner MAX plus the EP400 complex. Immunoprecipitation for MAX and EP400 complex components confirmed their association with ST. We determined that the ST-MYCL-EP400 complex binds together to specific gene promoters and activates their expression by integrating chromatin immunoprecipitation with sequencing (ChIP-seq) and RNA-seq. MYCL and EP400 were required for maintenance of cell viability and cooperated with ST to promote gene expression in MCC cell lines. A genome-wide CRISPR-Cas9 screen confirmed the requirement for MYCL and EP400 in MCPyV-positive MCC cell lines. We demonstrate that ST can activate gene expression in a EP400 and MYCL dependent manner and this activity contributes to cellular transformation and generation of induced pluripotent stem cells

MAX, EP400 and MCPyV ST bind to actively transcribed promoters.

<p>(A) Venn diagram of annotated genes corresponding to peaks identified by ChIP-seq with indicated antibodies. Two biological replicas of MAX and EP400 were performed and shared genes indicated. Shared genes identified with Ab5 and ST-HA are indicated. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006668#ppat.1006668.s006" target="_blank">S6A Fig</a>. (B) De novo DNA motif identification with indicated antibodies. (C) Distribution of peaks by Metagene analysis. (D) Heatmaps of H3K4me3, MAX, EP400 and ST (Ab5) ChIP peaks ranked by read density of H3K4me3 and scaled against the 75th percentile of genome-wide read density for each ChIP. (E) Meta-track analysis of ChIP-seq read density for MAX, EP400 and ST at all H3K4me3 peaks genome-wide. Regions are centered and ranked for H3K4me3 peaks over input.</p

MCPyV ST cooperates with MYCL and EP400 complex to activate gene expression.

<p>(A) Heatmap depicts average mean-centered and standard-deviation-scaled gene expression profiles for each of 62 clusters created by applying model-based clustering to the differentially expressed genes (DEG) in MKL-1 cells after depletion of EP400 or MYCL in comparison to shScr control. Model-based clusters (1–62) are labeled on the right-hand side and their gene members are listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006668#ppat.1006668.s012" target="_blank">S3 Table</a>. Merged Clusters (CL1-4) are indicated on the left-hand side. (B) Diagram illustrating BETA Activating/Repressing Function Prediction of transcription factors by correlation of distance of peaks from corresponding TSS obtained in ChIP-seq of ST, MAX and EP400 with changes in gene expression by RNA-seq after Dox-induction with shRNA targeting EP400 or MYCL. (C) Venn diagram showing common direct target genes of MAX, EP400 and ST identified by BETA based on ChIP-seq of MAX, EP400, ST and RNA-seq of shEP400–2, -3 and MYCL shRNA (BETA3). (D) Heatmap depicts average mean-centered and standard-deviation-scaled gene expression profiles for each of 37 clusters created by applying model-based clustering to the 951 BETA3 target genes in MKL-1 cells after depletion of EP400 or MYCL in comparison to shScr control. Model-based clusters (1–37) are labeled on the right-hand side and their gene members are listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006668#ppat.1006668.s012" target="_blank">S3 Table</a>. Merged Clusters (CL1-4) are indicated on the left-hand side. (E) MKL-1 cells containing Dox inducible shRNA for shScr, shMYCL or EP400 (shEP400-2, -3) were treated with dox for 5 days. Lysates were blotted with indicated antibodies. EP400 immunoprecipitations were blotted with EP400 antibody.</p