Time-to-Palpability.

<p>The length of time lapsed after initial cell line injection to detection of palpable tumors (~2mm x 2mm) is indicated for each of the four MCC cell lines tested (MKL-1, WaGa, MKL-2, and MS-1). </p

Kaplan-Meier curves of multiple MCC mouse xenograft models on different treatments.

<p><b>A</b>) Estimated survival means and 95% confidence intervals are reported along compressed survival summaries per cell line and treatment arm, where open circles correspond survival of individual mice. <b>B</b>) Mice with MKL-1 xenografts exhibit significantly prolonged survival (****P < 0.0001) on any of the three YM155 treatment groups (3-weeks at 2mg/kg = red; continuous treatment at 2mg/kg = purple; continuous treatment at 4mg/kg = orange) relative to saline treatment (green). Increasing the duration of YM155 treatment from 3-weeks to continuous treatment at the 2mg/kg dose significantly prolongs survival (****P < 0.0001). Increasing the dose of YM155 from 2mg/kg to 4mg/kg on continuous treatment significantly prolongs survival (****P < 0.0001). <b>C</b>) Mice with MS-1 xenografts do not exhibit prolonged survival with YM155 continuous treatment (either at 2mg/kg or 4mg/kg) relative to saline treatment (NS = not significant). One mouse on saline treatment spontaneously regressed for over 5-weeks and was euthanized early (as indicated by <b>x</b>). <b>D</b>) Mice with WaGa xenografts exhibit significantly prolonged survival (**P = 0.0034) with continuous YM155 treatment at 4mg/kg relative to saline treatment. <b>E</b>) Mice with MKL-2 xenografts exhibit significantly prolonged survival (****P < 0.0001) with continuous YM155 treatment at 4mg/kg relative to saline treatment. Two mice did not reach the final 20mm tumor dimension by day 105 and were euthanized early (as indicated by <b>##</b>). </p

Immunohistochemistry of MCV-LT in a MKL-1 xenograft primary tumor and a liver metastasis.

<p>Shown are paired hemotoxylin & eosin (H&E) stained slides and adjacent sections stained with CM2B4, the MCV-LT antibody (LT-IHC), in mice with MKL-1 xenografts: <b>A</b>) MKL-1 xenograft primary tumor, H&E; <b>B</b>) MKL-1 xenograft primary tumor, LT-IHC; <b>C</b>) MKL-1 xenograft liver metastasis, H&E; and <b>D</b>) MKL-1 xenograft liver metastasis, LT-IHC. MKL-1 cells contains nuclear staining of LT, consistent with an intact nuclear localization signal (NLS). Original magnification = 200X; insets = 600X. </p

Various chemotherapeutics combined with YM155 induce MCC cell death in an additive manner, <i>in</i><i>vitro</i>.

<p>CellTiter-GLO assays were performed using multiple MCC cell lines as well as the control primary human fibroblast, BJ. Corresponding dose-response curves are shown for the following chemotherapeutic agents and drug combinations: <b>A</b>) YM155; <b>B</b>) Bortezomib; <b>C</b>) Bortezomib + 3nM YM155; <b>D</b>) Docetaxel; <b>E</b>) Docetaxel + 3nM YM155; <b>F</b>) Etoposide; <b>G</b>) Etoposide + 3nM YM155 <b>H</b>) Topotecan; and <b>I</b>) Topotecan + 3nM YM155. </p

MCC mouse xenograft treatment groups and experimental outline.

<p><b>A</b>) NSG mice were subcutaneously injected in the right flank with 2x10⁷ MCV-positive, MCC cells (MKL-1, MS-1, WaGa, or MKL-2). <b>B</b>) NSG mice were monitored for palpable tumors (~2mm x 2mm) to determine start of treatment. <b>C</b>) Mice with palpable tumors were randomly assigned to either saline treatment, YM155 treatment for 3-weeks at 2mg/kg, YM155 continuous treatment at 2mg/kg, or YM155 continuous treatment at 4mg/kg. Each week of treatment consisted of a single intraperitoneal injection per day for 5 days, followed by 2 days of rest. </p

Mouse weights by treatment regimen.

<p>Average mouse weights with standard deviations are reported according to treatment regimen, where weights were normalized to day zero of treatment (100%): <b>A</b>) mouse weights on saline, continuous-treatment (green line); <b>B</b>) mouse weights on 2mg/kg YM155, continuous-treatment (purple line); and <b>C</b>) mouse weights on 4mg/kg YM155, continuous-treatment (orange line). Mouse weights were adjusted to remove the weight of tumors prior to normalization. Weights from mice with significant liver metastases were not included as metastatic-tumor weights could not be determined during the course of treatment. </p

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

MCPyV ST, MYCL and EP400 complex cooperate to reprogram and transform cells.

<p>(A) HFK-hTERT cells were transduced with Dox-inducible OCT4, SOX2 and KLF4 (P) and stably expressed MYCL, 3M or 4M MCPyV ST. Cells were treated with Dox for 31 days and then were immunostained with fluorescent antibodies to TRA-1-60 or TRA-1-81. Light field images demonstrate flat iPSC colonies formed with 3M and MYCL but not from 4M. (B) Cells were stained with alkaline phosphatase one day after immunostaining (Fig 5A). (C) Number of iPSC colonies detected after 31 days. Three biological replicas were performed. Data are presented as mean (SD). (D) IMR90 cells stably expressing dominant negative p53 and hTERT (PH) were transduced with MYCL (PHL) or tumor derived MCPyV ER region containing truncated LT and wild type ST (PHE) and MYCL (PHEL) or 3M mutant ST (PH3) and 4M mutant ST (PH4). Lysates (Input) were prepared from indicated cells, immunoprecipitated with Ab5 or MAX antibodies followed by immunoblotting with the indicated antibodies. (E) Images of soft agar colonies from PHEL cells (4X or 20X magnification). (F) Anchorage independent growth of IMR90 cells indicated in D (10⁵ cells) plated in soft agar and cultured for 4 weeks. Three biological replicas were performed. Data are presented as mean (SD).</p

MCPyV ST binds MYCL and EP400 complex.

<p>(A) MCPyV early region showing nucleotide positions for LT start (5387), ST stop (4827), LT stop (2503), and LT splice donor (5154) and acceptor (4722) and approximate positions of mutations that result in truncated LT found in MCC. LT and ST share an N-terminal J domain. The ST unique domain contains the LSD and Zn fingers. LT splices from J domain to a second exon containing the LXCXE or RB1 binding motif. Antibody Ab3 binds LT only and Ab5 binds both LT and ST. (B) Identification of co-precipitating proteins by MudPIT with antibodies Ab3 (LT, blue), Ab5 (LT/ST, red), EP400 (green) and MAX (black). See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006668#ppat.1006668.s010" target="_blank">S1 Table</a> for details. (C) MKL-1 lysates were immunoprecipitated (IP) with indicated antibodies (top) followed by immunoblotting with indicated antibodies (left). Asterisks indicate non-specific bands in IgG control immunoprecipitation lane. (D) MKL-1 lysates (Input) were separated in a Superose 6 column and fractions (#) were blotted with antibodies indicated on left. Protein size markers in kDa indicated at top and right. (E) Three MYCL isoforms (i1, i2, i3) are indicated (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006668#ppat.1006668.s001" target="_blank">S1 Fig</a>). Immunogen of MYCL antibody contained MYCL-i1 residues 16–139. (F) Fractions #5, 13 and 21 from Fig 1D were immunoprecipitated with MAX antibody and blotted.</p