MERKEL CELL POLYOMAVIRUS POSITIVE MERKEL CELL CARCINOMA - FROM DIAGNOSIS TO THERAPY

Merkel cell polyomavirus (MCV) is one of the newer members of the polyomavirus family, recently discovered as clonally integrated into the genomes of a subset of Merkel cell carcinoma (MCC). MCV is the first polyomavirus that is widely accepted to cause a human cancer and its identification has resulted in a paradigm shift in the understanding of MCC biology. In the vast majority of the population, MCV is a harmless member of the normal human microbial flora, but can initiate an aggressive cancer if it integrates into the host genome and acquires a precise set of viral mutations that result in replication incompetence (in a susceptible host). This dissertation describes how the identification of a new viral cause of MCC was harnessed to develop new diagnostic assays and therapeutic options for MCC. To assess MCV infection and its association with MCC as well as other human diseases, a monoclonal antibody that specifically recognizes both endogenous and transfected MCV large T antigen was developed. Using this antibody, specific nuclear localization of MCV T antigen in MCC tumor cells was demonstrated. A quantitative PCR assay revealed that MCV is present in MCC at >1 copy per tumor cell. These assays were used to survey non-MCC tissues including hematolymphoid malignancies, neuroendocrine tumors, and various dermatologic cancers.None showed association with MCV infection. Restricted expression of the MCV LT oncoprotein to MCC tumor cells provides the mechanistic underpinning supporting the notion that MCV causes a subset of MCC. To investigate treatment options for MCC two methods were used. An in vitro drug screen of 1360 chemotherapeutic and pharmacologically active compounds resulted in the identification of a proteasome inhibitor, bortezomib, as a potent but nonselective candidate. To rationally and specifically target MCV positive MCC, deep-sequence profiles of MCV positive MCC tumors were compared to MCV negative MCC tumors. Among 64 cell death related genes, a seven fold differential expression of survivin was observed in MCV positive MCC. MCV T antigen knock down in MCV positive cell lines decreased survivin mRNA and protein expression. Also, exogenously expressed MCV T antigen increased survivin protein in non-MCC primary cells in an RB protein binding dependent manner. A survivin inhibitor, YM155 initiated selective nonapoptotic MCV positive MCC cell death. YM155 was nontoxic and halted MCV positive MCC xenograft tumor growth in mice while bortezomib was inactive and caused significant toxicity in vivo

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

Merkel Cell Polyomavirus in Merkel Cell Carcinoma: Integration Sites and Involvement of the KMT2D Tumor Suppressor Gene

Merkel cell carcinoma (MCC) is an uncommon, lethal cancer of the skin caused by either Merkel cell polyomavirus (MCPyV) or UV-linked mutations. MCPyV is found integrated into MCC tumor genomes, accompanied by truncation mutations that render the MCPyV large T antigen replication incompetent. We used the open access HPV Detector/Cancer-virus Detector tool to determine MCPyV integration sites in whole-exome sequencing data from five MCC cases, thereby adding to the limited published MCPyV integration site junction data. We also systematically reviewed published data on integration for MCPyV in the human genome, presenting a collation of 123 MCC cases and their linked chromosomal sites. We confirmed that there were no highly recurrent specific sites of integration. We found that chromosome 5 was most frequently involved in MCPyV integration and that integration sites were significantly enriched for genes with binding sites for oncogenic transcription factors such as LEF1 and ZEB1, suggesting the possibility of increased open chromatin in these gene sets. Additionally, in one case we found, for the first time, integration involving the tumor suppressor gene KMT2D, adding to previous reports of rare MCPyV integration into host tumor suppressor genes in MCC

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

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

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

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

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

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

Methyltransferase-independent function of enhancer of zeste homologue 2 maintains tumorigenicity induced by human oncogenic papillomavirus and polyomavirus

Merkel cell polyomavirus (MCV) and high-risk human papillomavirus (HPV) are human tumor viruses that cause Merkel cell carcinoma (MCC) and oropharyngeal squamous cell carcinoma (OSCC), respectively. HPV E7 and MCV large T (LT) oncoproteins target the retinoblastoma tumor suppressor protein (pRb) through the conserved LxCxE motif. We identified enhancer of zeste homolog 2 (EZH2) as a common host oncoprotein activated by both viral oncoproteins through the pRb binding motif. EZH2 is a catalytic subunit of the polycomb 2 (PRC2) complex that trimethylates histone H3 at lysine 27 (H3K27me3). In MCC tissues EZH2 was highly expressed, irrespective of MCV status. Loss-of-function studies revealed that viral HPV E6/E7 and T antigen expression are required for Ezh2 mRNA expression and that EZH2 is essential for HPV(+)OSCC and MCV(+)MCC cell growth. Furthermore, EZH2 protein degraders reduced cell viability efficiently and rapidly in HPV(+)OSCC and MCV(+)MCC cells, whereas EZH2 histone methyltransferase inhibitors did not affect cell proliferation or viability within the same treatment period. These results suggest that a methyltransferase-independent function of EZH2 contributes to tumorigenesis downstream of two viral oncoproteins, and that direct targeting of EZH2 protein expression could be a promising strategy for the inhibition of tumor growth in HPV(+)OSCC and MCV(+)MCC patients