T antigens inhibit Notch to promote Merkel cell carcinoma in skin stem cells

项目摘要 默克尔细胞癌（MCC）是一种高度侵袭性的皮肤神经内分泌恶性肿瘤。一个新 发现的人类多瘤病毒，默克尔细胞多瘤病毒（MCV），编码致癌的小和大T 在80%以上的MCC中发现的抗原。MCV T抗原促进正常细胞的转化， 用于MCC维护。但是这些病毒蛋白质的精确分子机制 实现这一点还有待确定。研究MCV T抗原将提供一个独特的机会， 研究MCC和其他侵袭性神经内分泌癌的发病机制， 需要的治疗目标。最近，DeCaprio实验室发现MCV小T抗原（ST） 与MYCL（L-MYC）转录因子和EP 400染色质重塑复合物特异性结合， 激活基因表达。我们发现ST与EP 400和MYCL的复合物激活了默克尔细胞- 特异性转录因子ATOH 1，以及corREST转录阻遏物复合物的组分。 在MCC中使用化学抑制剂的coREST失活导致生长停滞和Notch肿瘤的增加 信号抑制基因在这里，我们假设，通过直接激活和间接抑制， Notch信号传导基因通过coREST、ST增加MCC中的ATOH 1以促进肿瘤发生。我们还发现 coREST失活上调LGR 6皮肤干细胞受体。体内LGR 6表达受限 通常是毛囊干细胞。最近在LGR 6谱系追踪小鼠模型中的研究表明，LGR 6 + 成体干细胞能够在创伤后重建整个滤泡间上皮，这表明 显示默克尔细胞特性的MCC可能来自这些细胞。因此，我们建议 以下具体目的是检验默克尔细胞多瘤病毒通过以下方式引起癌症的假设： 在毛囊干细胞中激活ATOH 1和抑制Notch。在目标1中，我将确定ST是否 通过直接激活EP 400-MYCL和通过coREST抑制Notch信号传导基因来诱导ATOH 1。我会 研究ST在MCC细胞系中的占据以及coREST对Notch信号基因表达的影响。在 目的2，我将评估ATOH 1转录程序和Notch信号在MCC维持中的作用。到 因此，我将研究ATOH 1转录组，并探讨Notch信号激活在MCC中的作用。在Aim中 3、我建议确定LGR 6是否驱动默克尔细胞中多瘤病毒T抗原的表达 毛囊干细胞导致小鼠皮肤中ATOH 1+默克尔细胞样细胞的积累。 影响：在完成这些目标后，拟议的研究将揭示ATOH 1未被重视的作用， 研究ST如何通过调节COREST和Notch信号通路在MCC中的作用， 主机转录程序。这些发现将揭示MCC和其他潜在的癌症靶点。 难以治疗的神经内分泌恶性肿瘤。

Project Summary Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine malignancy in the skin. A newly discovered human polyomavirus, Merkel cell polyomavirus (MCV), encodes oncogenic small and large T antigens that are found in more than 80% of MCC. MCV T antigens promote transformation of normal cells and are required for MCC maintenance. But the precise molecular mechanism by which these viral proteins accomplish this has yet to be determined. Studying MCV T antigens will provide a unique opportunity to investigate the pathogenesis of MCC and other aggressive neuroendocrine cancers and to discover much needed therapeutic targets. Recently, the DeCaprio laboratory discovered that MCV Small T antigen (ST) specifically associates with MYCL (L-MYC) transcription factor and the EP400 chromatin remodeler complex to activate gene expression. We found that ST in a complex with EP400 and MYCL activates the Merkel cell- specific transcription factor ATOH1, as well as components of the coREST transcription repressor complex. coREST inactivation using chemical inhibitors in MCC causes a growth arrest and an increase in Notch tumor suppressor signaling genes. Here we hypothesize that through direct activation and indirect repression of Notch signaling genes via coREST, ST increases ATOH1 in MCC to promote oncogenesis. We also found that coREST inactivation upregulates the LGR6 skin stem cell receptor. In vivo LGR6 expression is restricted normally to hair follicle stem cells. Recent work in an LGR6 lineage-tracing mouse model suggests that LGR6+ adult stem cells are able to reconstitute the entire interfollicular epithelium after wounding, suggesting the possibility that MCC displaying Merkel cell properties may arise from these cells. Thus, we propose the following specific aims to test the hypothesis that Merkel cell polyomavirus causes cancer by activating ATOH1 and inhibiting Notch in hair follicle stem cells. In Aim 1, I will determine whether ST induces ATOH1 through direct activation EP400-MYCL and Notch signaling gene repression by coREST. I will investigate ST’s occupancy and the effect of coREST on Notch signaling gene expression in MCC cell lines. In Aim 2, I will assess the role of the ATOH1 transcription program and Notch signaling in MCC maintenance. To do so, I will study the ATOH1 transcriptome and probe the effect of Notch signaling activation in MCC. In Aim 3, I propose to determine whether LGR6-driven expression of Merkel cell polyomavirus T antigens in the hair follicle stem cells leads to accumulation of ATOH1+ Merkel cell-like cells in mouse skin. Impact: After completing these aims, the proposed research will uncover the unappreciated role of ATOH1, coREST and Notch signaling pathway in MCC and determine how ST contributes to MCC through modulating host transcription programs. These findings will disclose novel cancer targets for MCC and potentially other difficult to treat neuroendocrine malignancies.