Rev1 overexpression accelerates N-methyl-N-nitrosourea (MNU)-induced thymic lymphoma by increasing mutagenesis

Abstract

Rev1 has two important functions in the translesion synthesis pathway, including dCMP transferase activity, and acts as a scaffolding protein for other polymerases involved in translesion synthesis. However, the role of Rev1 in mutagenesis and tumorigenesis in vivo remains unclear. We previously generated Rev1-overexpressing (Rev1-Tg) mice and reported that they exhibited a significantly increased incidence of intestinal adenoma and thymic lymphoma (TL) after N-methyl-N-nitrosourea (MNU) treatment. In this study, we investigated mutagenesis of MNU-induced TL tumorigenesis in wild-type (WT) and Rev1-Tg mice using diverse approaches, including whole-exome sequencing (WES). In Rev1-Tg TLs, the mutation frequency was higher than that in WT TL in most cases. However, no difference in the number of nonsynonymous mutations in the Catalogue of Somatic Mutations in Cancer (COSMIC) genes was observed, and mutations involved in Notch1 and MAPK signaling were similarly detected in both TLs. Mutational signature analysis of WT and Rev1-Tg TLs revealed cosine similarity with COSMIC mutational SBS5 (aging-related) and SBS11 (alkylation-related). Interestingly, the total number of mutations, but not the genotypes of WT and Rev1-Tg, was positively correlated with the relative contribution of SBS5 in individual TLs, suggesting that genetic instability could be accelerated in Rev1-Tg TLs. Finally, we demonstrated that preleukemic cells could be detected earlier in Rev1-Tg mice than in WT mice, following MNU treatment. In conclusion, Rev1 overexpression accelerates mutagenesis and increases the incidence of MNU-induced TL by shortening the latency period, which may be associated with more frequent DNA damage-induced genetic instability.

Keywords: DNA translesion synthesis; mutagenesis; thymic lymphoma; tumorigenesis; whole‐exome sequencing.

FIGURE 1

Thymic lymphoma (TL)‐free survival curves of WT (solid line) and Rev1‐Tg mice (dotted line) after repeated N‐methyl‐N‐nitrosourea (MNU) treatment by intraperitoneal injection. The hazard ratio (log‐rank) for WT/Rev1‐Tg was 0.4624 [0.2636–0.7259].

FIGURE 2

Mutational landscape of N‐methyl‐N‐nitrosourea (MNU)‐induced thymic lymphomas (TLs) in WT and Rev1‐Tg mice identified by whole‐exome sequencing (WES). Mutations are separated into single‐nucleotide variants (SNVs) and insertions and deletions (InDels). (A) The numbers of SNVs and InDels per TL sample from WT and Rev1‐Tg mice. (B) Frequency of each mutation according to the type of base substitution in each TL sample.

FIGURE 3

Contribution of the Catalogue of Somatic Mutations in Cancer (COSMIC) signatures to individual thymic lymphomas (TLs) from WT and Rev1‐Tg mice. (A) Heatmap with the cosine similarity between the mutational profile of each TL and its COSMIC signatures. (B) Absolute contribution of COSMIC SBS5 and SBS11 in each TL sample from WT and Rev1‐Tg mice.

FIGURE 4

Genetic alterations in the putative driver genes from N‐methyl‐N‐nitrosourea (MNU)‐induced WT and Rev1‐Tg thymic lymphomas (TLs) identified by whole‐exome sequencing (WES).

FIGURE 5

The number of Catalogue of Somatic Mutations in Cancer (COSMIC) Cancer Gene Census (CGC) genes with nonsynonymous mutations in each thymic lymphoma (TL).

FIGURE 6

T cell receptor (TCR) mutant assay. TCR mutant frequency in peripheral T cells from spleens of WT and Rev1‐Tg mice after N‐methyl‐N‐nitrosourea (MNU) treatment. The numbers of mice used are indicated in brackets below the x‐axis.

FIGURE 7

Clonal growth of thymocytes in WT and Rev1‐Tg mice after N‐methyl‐N‐nitrosourea (MNU) treatment. (A) Schematic illustration of D‐J rearrangement site and primer set in the TCRβ locus. (B) D‐J rearrangement pattern in the TCRβ locus in thymocytes after MNU treatment and in cells from MNU‐induced thymic lymphoma (TL).