Oncogenic Merkel Cell Polyomavirus T Antigen Truncating Mutations are Mediated by APOBEC3 Activity in Merkel Cell Carcinoma

Abstract

Merkel cell carcinoma (MCC) is an aggressive skin cancer, which is frequently caused by Merkel cell polyomavirus (MCPyV). Mutations of MCPyV tumor (T) antigens are major pathologic events of virus-positive (MCPyV+) MCCs, but their source is unclear. Activation-induced cytidine deaminase (AID)/APOBEC family cytidine deaminases contribute to antiviral immunity by mutating viral genomes and are potential carcinogenic mutators. We studied the contribution of AID/APOBEC cytidine deaminases to MCPyV large T (LT) truncation events. The MCPyV LT area in MCCs was enriched with cytosine-targeting mutations, and a strong APOBEC3 mutation signature was observed in MCC sequences. AICDA and APOBEC3 expression were detected in the Finnish MCC sample cohort, and LT expression correlated with APOBEC3H and APOBEC3G. Marginal but statistically significant somatic hypermutation targeting activity was detected in the MCPyV regulatory region. Our results suggest that APOBEC3 cytidine deaminases are a plausible cause of the LT truncating mutations in MCPyV+ MCC, while the role of AID in MCC carcinogenesis is unlikely.

Significance: We uncover APOBEC3 mutation signature in MCPyV LT that reveals the likely cause of mutations underlying MCPyV+ MCC. We further reveal an expression pattern of APOBECs in a large Finnish MCC sample cohort. Thus, the findings presented here suggest a molecular mechanism underlying an aggressive carcinoma with poor prognosis.

FIGURE 1

Mutations in MCV LT area in MCC and control samples. A, Substitution mutations in LT area in MCC and control samples in six mutation classes: C>A, C>T, C>G and T>A, T>C, T>G. Number of analyzed substitutions is shown in the middle. B, Distribution of substitutions along LT area is in MCC and control samples. LT area is divided to 200 bp bins (final bin 285 bp) and mutation frequency (number of mutations/number of sequences per bin) is shown. A schematic of ST and LT area is depicted below the graphs. C, Base content in LT area in each bin. D, Distribution of substitutions by mutated base in each bin. E, Distribution of in-frame stop codons introduced by substitution mutations in each bin (light gray). Black columns represent the proportion of in-frame stop codons that reside within APOBEC3 TCW hotspots. F, Distribution of in-frame stop codons in each bin introduced by insertion/deletion (indel) mutations before or in the indicated bin. Statistical significances between MCC and control mutations are determined using Mann–Whitney U test. **** <0.0001, *** <0.001, ** <0.01, * <0.05.

FIGURE 2

SHM targeting activity of MCV genome. A, Schematic of MCPyV genome and fragments tested in GFP loss assay. Arrows in “Fr1” and “Fr1 flipped” highlight the opposite orientation of these fragments relative to the GFP reporter (37). B, SHM targeting activity in MCPyV genome as measured in GFP loss assay. Four subregions (Fr1, Fr2, Fr3, and Fr4) of MCPyV genome as well as truncated Fr1, Fr2, and Fr1 in reverse orientation (Fr1 flipped) were tested (no CTCF). MCPyV NCCR (440 bp) and its 236, 80, and 204 bp subfragments were tested also downstream of the transcription unit (236bpD, 440bpD, 80bpD, and 204bpD). Human Ig lambda enhancer core (37) was used as positive control and the reporter without a test sequence was used as negative control. Median values are shown. Ten datapoints are outside of axis limits. Mann–Whitney U test was used to determine statistical significance. P values: pos. control <0.0001; Fr1 0.7863; Fr 2 0.4659; Fr3 0.0013; Fr4 0.7144; Fr1 truncated 0.9215; Fr2 truncated 0.7978; MCV Fr1 flipped 0.0901; 236bp 0.0009; 236bpD <0.0001; 440bp 0.0004; 440bpD 0.0161; 80bp 0.0672; 80bpD 0.4128; 204bp 0.0001; 204bpD 0.3783.

FIGURE 3

AID, APOBEC, and UV hotspot mutations in LT area. A, The proportion of substitutions in AID (WRC), APOBEC3 (TCW), and UV (YCC) hotspots in sequences from MCC (black) and control (gray) samples. B, Proportion of APOBEC3 subfamily hotspot mutations in YTCR, ATCR, WYCR, TC, TTCW, and CCCH in MCC samples and control samples. Colors as in A. C, The proportion of mutation type (C>T dark gray, C>G light gray and C>A black) in each hotspot in MCC samples. Number of each mutation type as well as total number of mutations in hotspots is indicated. D, The proportion of mutation type (C>T, C>G, and C>A) in each hotspot in control samples. Numbers and colors are as in C. E, The proportion of mutation type (C>T, C>G, and C>A) in each hotspot in MCC samples. Numbers and colors are as in C. F, The proportion of mutation type (C>T, C>G, and C>A) in each hotspot in control samples. Numbers and colors are as in C. G, Distribution of mutated WRC, TCW, and YCC hotspots along MCPyV LT area. Statistical significances between MCC and control hotspot mutations and their types are determined using Mann–Whitney U test. **** <0.0001, *** <0.001, ** <0.01, * <0.05.

FIGURE 4

AICDA and APOBEC expression in Finnish MCC sample cohort. A, Heatmap of AID/APOBEC expression across individual tumors. Hierarchical clustering was performed with one minus Spearman rank correlation method. MCC samples are arranged according to LT expression level. B, Similarity matrix of AICDA, APOBEC, and LT expression. APOBEC3H and APOBEC3G are significantly coexpressed with LT (Spearman rank correlation APOBEC3H r = 0.32, P = 0.004 and APOBEC3G r = 0.25, P = 0.025). C, Similarity matrix of selected T lymphocyte, B lymphocyte, NK-cell, and follicular dendritic cell markers and their coexpression with AICDA, APOBECs, and LT.