Response of Merkel cell polyomavirus-positive Merkel cell carcinoma xenografts to a survivin inhibitor

Merkel cell carcinoma (MCC) is a neuroendocrine skin cancer associated with high mortality. Merkel cell polyomavirus (MCV), discovered in 2008, is associated with ∼80% of MCC. The MCV large tumor (LT) oncoprotein upregulates the cellular oncoprotein survivin through its conserved retinoblastoma protein-binding motif. We confirm here that YM155, a survivin suppressor, is cytotoxic to MCV-positive MCC cells in vitro at nanomolar levels. Mouse survival was significantly improved for NOD-Scid-Gamma mice treated with YM155 in a dose and duration dependent manner for 3 of 4 MCV-positive MCC xenografts. One MCV-positive MCC xenograft (MS-1) failed to significantly respond to YM155, which corresponds with in vitro dose-response activity. Combination treatment of YM155 with other chemotherapeutics resulted in additive but not synergistic cell killing of MCC cell lines in vitro. These results suggest that survivin targeting is a promising therapeutic approach for most but not all MCV-positive MCCs. © 2013 Dresang et al

Coupled transcriptome and proteome analysis of human lymphotropic tumor viruses: insights on the detection and discovery of viral genes

<p>Abstract</p> <p>Background</p> <p>Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) are related human tumor viruses that cause primary effusion lymphomas (PEL) and Burkitt's lymphomas (BL), respectively. Viral genes expressed in naturally-infected cancer cells contribute to disease pathogenesis; knowing which viral genes are expressed is critical in understanding how these viruses cause cancer. To evaluate the expression of viral genes, we used high-resolution separation and mass spectrometry coupled with custom tiling arrays to align the viral proteomes and transcriptomes of three PEL and two BL cell lines under latent and lytic culture conditions.</p> <p>Results</p> <p>The majority of viral genes were efficiently detected at the transcript and/or protein level on manipulating the viral life cycle. Overall the correlation of expressed viral proteins and transcripts was highly complementary in both validating and providing orthogonal data with latent/lytic viral gene expression. Our approach also identified novel viral genes in both KSHV and EBV, and extends viral genome annotation. Several previously uncharacterized genes were validated at both transcript and protein levels.</p> <p>Conclusions</p> <p>This systems biology approach coupling proteome and transcriptome measurements provides a comprehensive view of viral gene expression that could not have been attained using each methodology independently. Detection of viral proteins in combination with viral transcripts is a potentially powerful method for establishing virus-disease relationships.</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

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

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

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

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

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