Reversal of viral and epigenetic HLA class I repression in Merkel cell carcinoma
Patrick C. Lee, et al.
Patrick C. Lee, et al.
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Research Article Oncology

Reversal of viral and epigenetic HLA class I repression in Merkel cell carcinoma

Abstract

Cancers avoid immune surveillance through an array of mechanisms, including perturbation of HLA class I antigen presentation. Merkel cell carcinoma (MCC) is an aggressive, HLA-I–low, neuroendocrine carcinoma of the skin often caused by the Merkel cell polyomavirus (MCPyV). Through the characterization of 11 newly generated MCC patient-derived cell lines, we identified transcriptional suppression of several class I antigen presentation genes. To systematically identify regulators of HLA-I loss in MCC, we performed parallel, genome-scale, gain- and loss-of-function screens in a patient-derived MCPyV-positive cell line and identified MYCL and the non-canonical Polycomb repressive complex 1.1 (PRC1.1) as HLA-I repressors. We observed physical interaction of MYCL with the MCPyV small T viral antigen, supporting a mechanism of virally mediated HLA-I suppression. We further identify the PRC1.1 component USP7 as a pharmacologic target to restore HLA-I expression in MCC.

Authors

Patrick C. Lee, Susan Klaeger, Phuong M. Le, Keegan Korthauer, Jingwei Cheng, Varsha Ananthapadmanabhan, Thomas C. Frost, Jonathan D. Stevens, Alan Y.L. Wong, J. Bryan Iorgulescu, Anna Y. Tarren, Vipheaviny A. Chea, Isabel P. Carulli, Camilla K. Lemvigh, Christina B. Pedersen, Ashley K. Gartin, Siranush Sarkizova, Kyle T. Wright, Letitia W. Li, Jason Nomburg, Shuqiang Li, Teddy Huang, Xiaoxi Liu, Lucas Pomerance, Laura M. Doherty, Annie M. Apffel, Luke J. Wallace, Suzanna Rachimi, Kristen D. Felt, Jacquelyn O. Wolff, Elizabeth Witten, Wandi Zhang, Donna Neuberg, William J. Lane, Guanglan Zhang, Lars R. Olsen, Manisha Thakuria, Scott J. Rodig, Karl R. Clauser, Gabriel J. Starrett, John G. Doench, Sara J. Buhrlage, Steven A. Carr, James A. DeCaprio, Catherine J. Wu, Derin B. Keskin

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Figure 5

The PRC1.1 complex implicated as a suppressor of HLA-I in a genome-wide CRISPR screen.

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The PRC1.1 complex implicated as a suppressor of HLA-I in a genome-wide ...
(A) Gene-level ranking of positive (left) and negative (right) CRISPR-KO screen hits, according to STARS, a gene-ranking algorithm for genetic screens (39). Inset: GSEA analysis of screen hits. (B) Flow cytometry for surface HLA-I in MCC-301 PRC1.1 KO lines (PCGF1, USP7, and BCORL1). Data visualized with biexponential scaling. (C) Western blot for PCGF1 (top) and USP7 (bottom) in WT MCC-301, a control sgRNA MCC-301 line, or the indicated knockout line. (D) Top: Volcano plot showing LFC in gene expression in an MCC-301 PCGF1-KO line compared with a control sgRNA line. Bottom: GSEA plot demonstrating enrichment of PRC2 target genes upon PCGF1 knockout. (E) Western blot of TAP1 in PCGF1-KO and control sgRNA lines at varying IFN-γ concentrations. (F) RNA-Seq analysis of HLA-I genes, PRC1.1, PRC2, and ST-MYCL-EP400 in a cohort of 51 MCC tumors. Left: Unsupervised hierarchical clustering heatmap by Euclidian distance. Top track: Tumor purity scores for each tumor, generated by ESTIMATE (53). Pearson’s correlation coefficients between each PRC2, PRC1.1, or ST-MYCL-EP400 component and each class I gene were calculated, and the bar charts (right) show the number of Pearson’s coefficients that were less than –0.3. (G) UCSC Genome Browser view of USP7 and PCGF1 with ChIP-Seq tracks for MAX (red), EP400 (blue), MCPyV ST antigen (pink), and activating histone marks H3K4me3 and H3K27ac (black). The “-1” and “-2” suffixes refer to 2 different antibodies used for each protein. (H) ChIP-qPCR targeting the USP7 and PCGF1 promoters, using MKL-1 chromatin immunoprecipitated with a MAX (left) or EP400 (right) antibody (n = 3). P values were calculated by 1-way ANOVA followed by post hoc Dunnett’s multiple-comparison test. (I) Protein expression of USP7, PCGF1, and MYCL in MKL-1 cells transduced with the indicated doxycycline-inducible shRNAs. (J) Schematic of putative interactions between MCPyV viral antigens and screen hits MYCL and PRC1.1.