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.

Rev1 overexpression accelerates mutagenesis and increases the incidence of N‐methyl‐N‐nitrosourea (MNU)‐induced thymic lymphoma (TL) by shortening the latency period, which may be associated with more frequent DNA damage‐induced genetic instability.

Abbreviations

CGC

Cancer Gene Census

CGH

comparative genomic hybridization

CNV(s)

copy number variant(s)

COSMIC

Catalogue of Somatic Mutations in Cancer

InDel(s)

insertion(s) and deletion(s)

MNU

N‐methyl‐N‐nitrosourea

PEST

polypeptide enriched in praline, glutamate, serine, and threonine

SNV(s)

single‐nucleotide variant(s)

TCR

T cell receptor

TL

thymic lymphoma

WES

whole‐exome sequencing

WT

wild type

1. INTRODUCTION

Mutagenic replication of damaged DNA via translesion synthesis is involved in the accumulation of mutations that lead to cancer development. ¹ , ² , ³ , ⁴ Translesion synthesis is known as the damage tolerance pathway because it promotes the bypassing of DNA lesions, including thymine dimers and abasic sites, thus overcoming replication blockage and promoting cell survival. Rev1 is a central player in translesion synthesis, and Rev1 deficiency is associated with an increased sensitivity to a wide range of DNA‐damaging agents. ⁵ , ⁶ , ⁷ We and others have previously investigated the biochemical properties of Rev1 and observed that human Rev1 displayed deoxycytidyl transferase activity, which incorporated deoxycytidines opposite guanines and DNA lesions such as abasic sites. ⁸ , ⁹ , ¹⁰ , ¹¹ , ¹² , ¹³ , ¹⁴ However, this catalytic activity has been found to be dispensable for vertebrate cell survival after exposure to DNA‐damaging agents. ¹⁵ , ¹⁶ Additionally, Rev1 interacts with multiple DNA polymerases involved in translesion synthesis through its C‐terminal domain. ¹⁷ , ¹⁸ , ¹⁹ , ²⁰ The C‐terminal domain of Rev1 is essential for its regulatory role as a molecular scaffold in translesion synthesis. ²¹ , ²² , ²³ Thus, the diverse functions of Rev1 determine its key role in maintaining genome integrity in the presence of DNA lesions, while concurrently promoting mutagenesis.

Altered Rev1 expression also influences DNA damage‐induced cellular toxicity. A human ovarian carcinoma cell line engineered to overexpress Rev1 exhibited increased resistance to the cytotoxic effects of cisplatin along with an increased frequency of cisplatin‐induced mutations. ²⁴ Moreover, Rev1 expression has been shown to predict radiotherapy sensitivity. ²⁵ Further findings have been reported regarding the function of Rev1 in vivo, such as that the suppression of Rev1 expression leads to reduced carcinogen‐induced lung tumors and inhibits the acquisition of tumor chemoresistance. ²⁶ , ²⁷ , ²⁸ , ²⁹ , ³⁰ , ³¹ , ³² Although the regulation of Rev1 expression is required to maintain genome integrity, and its dysregulation results in altered DNA damage‐induced cytotoxicity and mutagenicity, the general role of Rev1 in mutagenesis and carcinogenesis remains largely unexplored.

We previously generated Rev1‐Tg mice and reported a significantly increased incidence of small intestinal adenoma and thymic lymphoma (TL) after N‐methyl‐N‐nitrosourea (MNU) treatment in Rev1‐Tg male mice. ³³ Analysis of intestinal adenomas showed the direct dependency of mutagenicity and tumorigenicity on Rev1 expression levels, but no analysis was performed for TLs. Murine TL is a classic model for chemical or radiation‐induced tumorigenesis. ³⁴ , ³⁵ , ³⁶ , ³⁷ Recently, it has been reported that the application of high‐throughput sequencing identified key oncogenic drivers to gain insights into the mechanism of carcinogenesis, including radiation‐induced TL. ³⁸ , ³⁹ Next‐generation sequencing technology enables detailed observation of the DNA‐sequence‐level effects of well‐known mutagens, such as alkylating agents and ultraviolet radiation. Based on a preliminary small‐scale carcinogenesis study where we found that Rev1‐Tg female mice developed TL more frequently than WT mice and also more frequently than Rev1‐Tg male mice, here we used heterozygous Rev1‐Tg female mice to gain insights into the role of Rev1 overexpression in MNU‐induced mutagenesis and carcinogenesis in the thymus using whole‐exome sequencing (WES). We observed that Rev1 overexpression accelerated mutagenesis with no difference in the targeted driver genes, resulting in an increased incidence and shortened latency period for MNU‐induced TL. In our opinion, this is a valuable study investigating the role of Rev1 expression in the maintenance of genomic integrity in vivo.

2. MATERIALS AND METHODS

2.1. Mice and tumor induction

Female WT mice (C57BL/6 N) were purchased from Charles River. Rev1‐Tg mice were established in our laboratory as described previously. ³³ In order to induce the development of TL, both Rev1‐Tg and WT female mice were injected intraperitoneally, once a week for 2 weeks beginning at 6 weeks of age, with MNU (Sigma) at 50 mg/kg of body weight—a concentration that has been previously established as appropriate in TL carcinogenesis studies. ⁴⁰ The mice were sacrificed under anesthesia at moribund points. Thymic tissues were removed, weighed, and then frozen in liquid nitrogen or fixed in formalin for routine histological examination. Enlarged thymus tissues weighed more than 50 mg, about 2.5 times the normal weight. Histopathological analysis of enlarged thymus tissues revealed malignant TL characterized by a “starry sky” pattern with numerous mitoses. ⁴¹ Furthermore, some enlarged thymus tissues were diagnosed by using FACS, where we observed an altered CD4/CD8 pattern accompanied by the proliferation of CD4+CD8+ double‐positive thymocytes and/or CD4–CD8+ single‐positive thymocytes. All the experiments were approved by the Ethics Committee of Hiroshima University (permission no. A21‐27) and performed according to the Guidelines for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Science, Hiroshima University.

2.2. Quantitative real‐time RT‐PCR

We performed the extraction of total RNA, reverse transcription to cDNA, and real‐time fluorescence quantitative PCR, as described previously. ³³ The primers used are listed in Table S1.

2.3. Whole‐exome sequencing analysis

For WES, genomic DNA from TLs or from normal ears were extracted using a DNeasy Blood & Tissue Kit (Qiagen). Exome sequencing was performed by Macrogen Japan. Paired‐end reads were aligned to the mouse reference sequence mm10 using the Burrows–Wheeler Aligner. Multiple mapped read pairs with identical external coordinates were collapsed to remove potential PCR duplicates using the Picard MarkDuplicate. Local realignment and mapping quality score recalibration were performed using the Genome Analysis Toolkit. SnpEff was used for the functional annotation of variants, exonic functions, and nonsynonymous variants, such as stop‐gain single‐nucleotide variants (SNVs), splicing, and frameshift indels. Known variants reported using the dbSNP142 database were excluded. We used the R package MutationalPatterns (version 1.4.2) (https://www.bioconductor.org/packages/release/bioc/html/MutationalPatterns.html) to extract and analyze the mutational spectrum of the tumors. Control‐FREEC software (version 10.8) was used to identify copy number changes in tumors compared with normal tissues. Segments that exhibited a copy number change that was statistically significant relative to normal ploidy were extracted (p values <0.05, Wilcoxon and Kolmogorov–Smirnov tests).

2.4. Sequencing of Notch1, Ikaros (Ikzf1), and Kras

For Notch1, a polypeptide enriched in the praline, glutamate, serine, and threonine (PEST) domain was sequenced using the primers listed in Table S1. The Ikzf1 exons 2–7 were sequenced using the primers shown in Table S1 (primers Ex‐2‐3‐F and Ex‐5‐6‐R, Ex6‐F2, and Ex7‐R4). Mutations in the splicing junction between exons 4 and 5 were detected by using the primers Itr‐4‐F and Itr‐4‐R (Table S1). For Kras, exons 2 and 3 were sequenced using the primers Kras‐2‐F and Kras‐2R (Table S1). Exon 1 was sequenced using the primers Kras‐G‐F and Kras‐G‐R (Table S1).

2.5. T cell receptor (TCR) mutant assay

TCR mutant frequency was determined as previously described. ⁴² Briefly, spleen cells were stained with fluorescein FITC‐labeled anti‐CD4 (CALTAG Laboratories), PE‐labeled anti‐I‐A/I‐E (BD Biosciences), and PE‐labeled anti‐CD3 antibodies (BD Biosciences). TCR mutant frequency values were calculated as the number of mutant (CD4+, I‐A/I‐E‐, CD3−) cells divided by the total number of CD4+ T cells using FACScan flow cytometer (Becton Dickinson).

2.6. Detection of clonally growing thymocytes by D‐J rearrangement patterns at TCR gene

DNA from the mouse thymus tissue was isolated using a DNeasy Blood & Tissue Kit (Qiagen). To determine the D‐J rearrangement patterns in the TCRβ locus, PCR was performed as described previously. ³⁷

2.7. Statistical analysis

Statistically significant differences and exact p‐values of comparisons between survival curves were determined using the log‐rank (Mantel–Cox) test using the GraphPad 6 software package. In all other cases, t‐tests or chi‐square tests were performed using the StatMate III and GraphPad 6 software packages.

3. RESULTS

3.1. Shortened latency period and higher incidence of MNU‐induced TL in Rev1‐Tg mice

To confirm our previous tumorigenesis study and to explore the effect of Rev1 overexpression on mutagenesis and carcinogenesis by focusing on MNU‐induced TL in a classic model, we investigated a heterozygous Rev1‐Tg female mouse line (T5 line). ³³ In many tissues, including the thymus, Rev1 mRNA expression was significantly increased in Rev1‐Tg mice compared with that in WT mice (Figure S1). After MNU treatment, we detected tumors in 100% of the mice used in this study, and TL was one of the tumor types with the highest incidence in both WT and Rev1‐Tg mice, in addition to small‐intestinal tumors and lymphoma. ³³ We confirmed that the average number of intestinal adenomas per mouse was significantly higher in Rev1‐Tg mice than in WT mice (Table S2). Both the incidence of TL (WT: 69.7%; Rev1‐Tg: 92.7%) and latency period differed significantly between WT and Rev1‐Tg mice (Figure 1 and Table S3). We confirmed the association between Rev1 overexpression and MNU‐induced TL development in an additional strain of Rev1‐Tg mice (T9 line) (Figure S2 and Table S3). These data confirmed that Rev1 overexpression was associated with increased susceptibility to chemically induced TL development.

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].

3.2. Analysis of somatic mutations and copy number variants (CNVs) in MNU‐induced WT and Rev1‐Tg TLs by performing WES

We performed WES on 18 age‐matched samples, including one WT normal thymus, eight WT TLs, and nine Rev1‐Tg TLs. While there was a large variation in the number of mutations between samples in TLs, the mean number of SNVs and insertions and deletions (InDels) was higher in Rev1‐Tg TLs than in WT TLs, that is, the mutation frequency was significantly higher in Rev1‐Tg TLs (3.18 × 10⁻⁴) than in WT TLs (1.40 × 10⁻⁴) (Figure 2A). The proportion of SNVs and InDels did not show statistically significant differences between the WT and Rev1‐Tg TLs; rather, the proportion of SNVs tended to increase as the total number of mutations increased in both WT and Rev1‐Tg TLs (Figure S3A). The majority of InDels manifested as 1‐bp insertions and 1‐bp deletions (Figure S4A). As the total number of SNVs increased, each type of InDel tended to increase in both WT and Rev1‐Tg TLs (Figure S4B).

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.

The mutation spectrum was dominated by C:G > T:A and T:A > C:G transitions, which was common among WT and Rev1‐Tg TLs (Figure 2B and Figure S3B). As the total number of mutations increased, the proportion of C:G > T:A in the total mutations tended to decrease for both WT and Rev1‐Tg TLs (Figure S3B). A linear relationship was also observed between the coefficients of C:G > T:A and T:A > C:G for both the WT and Rev1‐Tg TLs, with the intercept not passing through zero (Figure 2B and Figure S3C). Multinomial logistic regression was performed to explore factors associated with the mutation spectrum. The results obtained from the analysis showed that the mutation spectrum was more associated with the total number of mutations detected in TLs than genotypes such as WT and Rev1‐Tg (Table S4). These results suggest that the mutation pattern varies depending on the number of mutations per TLs, not the genotype.

To further characterize the mutational spectrum of the TLs, we performed mutational signature analysis as previously described and compared mutational signatures of mouse TLs with the Catalogue of Somatic Mutations in Cancer (COSMIC) mutational signatures of human cancers (Figure 3 and Figures S5 and S6). ⁴³ This analysis revealed the greatest similarity to the COSMIC signatures associated with aging and alkylating agents, namely, COSMIC SBS5 and SBS11, respectively (Figure 3A and Figures S5 and S6). Unsupervised hierarchical clustering of TLs based on data from mutation signatures showed that they tended to cluster according to the total number of mutations in the TL rather than the genotype of the TL (Figures 2 and 3A). The best correlation was found between the higher contribution of SBS5 mutations in TLs and a higher number of mutations, regardless of the genotype, as WT and Rev1‐Tg (Figure 3B and Figure S7). In contrast, Rev1 overexpression contributed little or slightly to SBS11 in MNU‐induced TLs (Figure 3B). Consequently, SBS5 mutations in TLs were well correlated with overexpression of Rev1 since TLs with a higher number of mutations were more frequently observed in Rev1‐Tg (8/9) than in WT (3/8) (Figure 3).

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.

To evaluate whether Rev1 overexpression‐induced carcinogenesis was caused by increased chromosomal instability, we analyzed numerical variations from WES data using the Control‐FREEC software for CNVs. The number of CNVs varied widely among all TLs, and there was no significant difference in the number of CNVs between WT and Rev1‐Tg TLs. We confirmed that there was no significant difference in the number of CNVs between WT and Rev1‐Tg TLs by using array comparative genomic hybridization (CGH) (Table S5). Our data suggest that the Rev1 overexpression‐induced acceleration of TL development was mediated by accelerated induction of mutations, but not by alterations in the genome copy numbers, with no difference in the mutation pattern among WT and Rev1‐Tg TLs.

3.3. Identification of putative genetic drivers of thymic lymphomagenesis in WT and Rev1 ‐Tg mice after MNU treatment

We examined individual COSMIC Cancer Gene Census (CGC) genes to investigate the genetic alterations that could contribute to the tumorigenesis of TLs. ³⁸ , ⁴⁴ We detected nonsynonymous somatic mutations in the pathways including Notch signaling, MAPK signaling, genome maintenance, and epigenetic modifiers (Figure 4). There was no difference in the number of nonsynonymous mutations and the mutation spectrum detected in the COSMIC CGC genes for WT and Rev1‐Tg TLs (Figures 4 and 5 and Figure S8). Approximately 88% (7/8) of WT TLs and 67% (6/9) of Rev1‐Tg TLs harbored nonsynonymous mutations in Notch1, especially in exon 34, which encodes the PEST domain (Table S6). We also detected nonsynonymous mutations in Ikzf1 and Kras genes at high frequencies in both WT and Rev1‐Tg TLs.

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).

Given the small sample size of MNU‐induced TLs that we analyzed by WES, we examined mutations in Notch1 (PEST), Ikzf1, and Kras by Sanger sequencing of cDNAs from additional 12 WT TLs and 25 Rev1‐Tg TLs. Nonsynonymous mutations in these genes were detected at a high frequency of 58% (7/12) in WT TLs and 84% (21/25) in Rev1‐Tg TLs (Table S7). The mutation spectrum was dominated by C:G > T:A, which was common among the WT and Rev1‐Tg TLs (Figure S9). Collectively, our analysis revealed common genetic alterations in MNU‐induced TLs in both WT and Rev1‐Tg mice.

Ikaros inactivation in murine TL can occur through various mechanisms, including point mutations and expression of splicing variants. ³⁴ , ³⁶ Among the WT samples, only one out of 20 (5%) expressed the Ikaros splicing variants of Ik‐8 and Ik‐9. Analysis of Rev1‐Tg mice revealed that seven out of 34 (21%) TLs exhibited splicing variants and reduced size bands (Figures S10 and S17). Consistent with a previous study, we detected a point mutation in the splicing donor site within Ikaros intron 4 from two TLs (TL58 and TL64), which might impair donor function during pre‐mRNA splicing, resulting in reduced size bands of Ikzf1 (Figure S11). Our data suggested that there were slight but nonsignificant differences in the frequency of splicing variants between WT and Rev1‐Tg TLs.

In conclusion, our findings demonstrate that MNU‐induced TLs exhibit a high frequency of nonsynonymous mutations in Notch1 and Ikzf1 regulating the Notch1 signaling pathway, and Kras, a member of the MAPK signaling pathway, both in WT and Rev1‐Tg mice.

3.4. T cell receptor somatic mutant frequency in WT and Rev1 ‐Tg mice following MNU treatment

We measured TCR mutant frequency in splenic T cells, to evaluate the mutability of peripheral somatic cells following MNU treatment in vivo. ⁴⁵ MNU treatment resulted in increased TCR mutant frequency in both WT and Rev1‐Tg mice, peaking at 2 weeks after the injection (Figure 6). Statistical analysis with Student's t‐test revealed that TCR mutant frequency was significantly higher in Rev1‐Tg mice (8.96 × 10⁻⁴) than in WT mice (7.20 × 10⁻⁴) (p = 0.007) 2 weeks after MNU treatment as well as 4 weeks after MNU treatment (WT: 3.17 × 10⁻⁴; Rev1‐Tg mice: 4.23 × 10⁻⁴; p = 0.002). TCR mutant frequency returned to basal levels in WT mice 7 weeks after MNU treatment but remained elevated in some Rev1‐Tg mice. We further confirmed the increased mutability phenotype using a second strain of Rev1‐Tg mice (T9 line) (Figure S12). These results indicated that the increased mutability in splenic peripheral T cells from Rev1‐Tg mice may be related to the increased cancer risk from MNU exposure.

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.

3.5. Emergence of clonally growing premalignant thymocytes in Rev1 ‐Tg and WT mice following MNU treatment

To identify the early processes leading to TL development, we analyzed D‐J rearrangement at the TCR β‐chain (TCRβ) gene locus and the expression patterns of cell surface markers. Genomic DNA from normal thymocytes of WT and Rev1‐Tg mice exhibited multiple bands resulting from rearrangements between different Dβ and Jβ positions (Figure 7, without MNU treatment, and Figure S16). In contrast, DNA from MNU‐induced TLs showed only one or a few bands, indicating that TL cells were of monoclonal (one prominent band) or oligoclonal (a few prominent bands) origin. Although the thymus weight did not differ between WT and Rev1‐Tg mice, one of 12 in WT and three of 12 in Rev1‐Tg mice exhibited clonal expansion 4 weeks after MNU treatment. Moreover, clonal growth was observed in four of seven WT and seven of seven Rev1‐Tg mice 7 weeks after MNU treatment (Figure 7). The flow cytometry analysis exhibited that the presence of clonally growing thymocytes in either WT or Rev1‐Tg mice was accompanied by an altered CD4/CD8 pattern in most cases, whereas mice with a polyclonal origin of the TCRβ gene invariably showed a normal cell surface marker profile (Figure 7 and Figure S13). Interestingly, higher mRNA expression of Hes1 and Deltex1, transcriptional targets of Notch1, was detected in Rev1‐Tg samples 4 weeks after MNU treatment compared with that in WT samples (Figure S14). Thus, clonally expanding prelymphoma thymocytes and the common subsequent functional changes might emerge earlier and more frequently in Rev1‐Tg mice than in WT mice after MNU treatment.

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).

4. DISCUSSION

Translesion synthesis is involved in the generation of both spontaneous and DNA damage‐induced mutations, and Rev1 plays a central role in translesion synthesis. In this study, we revealed that 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 not associated with alterations in the genome copy numbers. Further investigations are needed to assess the possibility that Rev1 overexpression accelerates MNU‐induced TL via chromosomal instability associated with structural variations. Prelymphoma cells, which possess properties of self‐renewal and differentiation, were reported to be generated before TL development and could result in TL development after acquiring additional mutations. We observed that clonally growing populations of thymocytes appeared much earlier and with higher frequency in Rev1‐Tg mice, which may indicate an earlier onset of TL initiation and progression. We assume that the emergence of clonally growing populations of thymocytes may have been influenced by the presence of higher TCR mutant frequency in Rev1‐Tg mice at 7 weeks after MNU treatment. In conclusion, our data suggest that Rev1 overexpression could induce the emergence of prelymphoma cells earlier and with higher frequency after MNU treatment and may also facilitate the acquisition of additional mutations by the prelymphoma, consequently leading to TL development.

What is the origin of the accumulated mutations in Rev1‐Tg TLs? Mutation signature analysis showed that WT and Rev1‐Tg TLs exhibited cosine similarity with COSMIC mutational SBS5 and SBS11. COSMIC SBS11 exhibited a mutational pattern resembling that of alkylating agents, whereas COSMIC SBS5 is found in multiple cancer types and exhibits age‐related clock‐like behavior. ⁴⁶ We detected mutations at high frequency in putative drive genes such as Notch1, Ikzf1, and Kras, and the majority were C:G > A:T, followed by T:A > C:G mutations, with the same frequency and proportion between WT and Rev1‐Tg TLs. Importantly, C:G > A:T mutations were characteristic of both SBS5 and SBS11, and T:A > C:G mutations were predominant in SBS5. Therefore, we could not determine whether mutations in driver genes, such as Notch1, Ikzf1, and Kras, were generated directly by O ⁶ ‐me‐G lesion bypass in MNU‐induced TL development. Rev1 has two important functions in the translesion synthesis pathway, including dCMP transferase activity and acts as a scaffolding protein for other translesion synthesis polymerases. The incorporation of deoxycytidine opposite O ⁶ ‐meG by Rev1 leads to an accurate translesion synthesis; therefore, the catalytic activity of Rev1 does not result in mutations. Rev1 acts as a scaffold protein and plays a major role in mutagenesis. Previous studies have reported that pol δ can incorporate a nucleotide T opposite to an O ⁶ ‐meG lesion site, following which pol ζ completes the O ⁶ ‐me‐G‐induced C:G > T:A transition. ⁴⁷ , ⁴⁸ Rev1's established interaction with these polymerases could contribute, at least in part, to the C:G > T:A transitions. ⁴⁷ Since Rev1 overexpression had little or no effect on the extent of the contribution of SBS11, Rev1 overexpression could not stimulate the pol δ‐pol ζ pathway to bypass O ⁶ ‐meG, or if it did occur, only to a negligible extent. Our data imply that the acceleration of MNU‐induced tumor development by Rev1 overexpression could not be largely attributed to the enhancement of Rev1‐directed O ⁶ ‐me‐G mutagenesis.

In addition to COSMIC SBS11, SBS5 has a substantial contribution to MNU‐induced TL development in both WT and Rev1‐Tg mice. SBS5 was found in multiple cancer types and exhibited age‐related clock‐like behavior. ⁴⁶ Importantly, the number of total mutations, but not the genotype 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 TLs with a large number of mutations. 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. It has been reported that the loss of FHIT at the fragile FRA3B locus was correlated with the mutational SBS5 substitution rate in 6649 human cancer samples. ⁴⁹ The authors assumed that genome instability might be caused by age‐associated FHIT loss due to fragile site breakage throughout life, inducing SBS5 mutations that occur in most cancer types. In our study, the frequency of mutations and CNVs at fragile sites, such as Fhit, was not different between WT and Rev1‐Tg TLs; however, the alterations in cancer‐related genes associated with Fhit loss were higher in Rev1‐Tg TLs than in WT TLs (Table S8 and Figure S15). Thus, Rev1 overexpression‐enhanced genetic instability could be a pathway different from that of FHIT deficiency.

Recent findings revealed a novel function of Rev1 in replication gap suppression at the replication fork, distinct from its canonical lesion bypass and postreplication gap‐filling roles. ⁵⁰ , ⁵¹ , ⁵² Since oncogenes and genome instability that drive cancer development can induce replication stress, Rev1 overexpression might play the role in promoting carcinogenesis by suppressing the harmful DNA gaps that are generated from oncogene‐induced replication stress. Dysregulation and overexpression of Rev1 has been identified in various tumors. ⁵³ Oncogenic signaling has been reported to activate the translesion synthesis pathway via pol κ and Rad18, suggesting that translesion synthesis may play diverse roles in cancer adaptation. ⁵⁴ , ⁵⁵ Overall, these findings suggest that translesion synthesis‐mediated lesion bypass, enhanced mutability, and mitigation of replication stress may be involved in cancer development and adaptation, regardless of whether these phenomena are a cause of carcinogenesis or its consequences. Further studies are required to gain a deeper understanding of the mechanisms by which Rev1 is involved in DNA damage response and its role in carcinogenesis in individual organs.

AUTHOR CONTRIBUTIONS

Megumi Sasatani: Conceptualization; data curation; formal analysis; funding acquisition; investigation; methodology; project administration; resources; supervision; validation; visualization; writing – original draft; writing – review and editing. Yang Xi: Conceptualization; formal analysis; writing – original draft; writing – review and editing. Kazuhiro Daino: Formal analysis; methodology; writing – review and editing. Atsuko Ishikawa: Formal analysis; methodology; writing – review and editing. Yuji Masuda: Writing – original draft; writing – review and editing. Junko Kajimura: Formal analysis; writing – review and editing. Jinlian Piao: Formal analysis; writing – review and editing. Elena Karamfilova Zaharieva: Writing – original draft; writing – review and editing. Hiroaki Honda: Methodology; writing – review and editing. Guanyu Zhou: Formal analysis; writing – review and editing. Kanya Hamasaki: Methodology; writing – review and editing. Yoichiro Kusunoki: Methodology; writing – review and editing. Tsutomu Shimura: Writing – original draft; writing – review and editing. Shizuko Kakinuma: Methodology; writing – review and editing. Yoshiya Shimada: Methodology; writing – review and editing. Kazutaka Doi: Formal analysis; writing – review and editing. Tomoko Ishikawa‐Fujiwara: Methodology; writing – review and editing. Yusuke Sotomaru: Methodology; resources; writing – review and editing. Kenji Kamiya: Conceptualization; project administration; writing – original draft; writing – review and editing.

FUNDING INFORMATION

This work was supported by Japan Society for the Promotion of Science, JSPS KAKENHI (22310037 to KK, 20710043 to MS, 22710055 to MS). This work was also supported in part by the Nuclear Energy S&T and Human Resource Development Project (JPMX08S08080294) to MS, and the National Institute for Fusion Science, Collaborative Research Program (NIFS10KOBS015, NIFS13KOBA028, NIFS20KOCA004) to MS, and in part by the Initiative for Realizing Diversity in the Research Environment (Specific Correspondence Type), a support project for the Development of Human Resources in Science and Technology conducted by the Ministry of Education, Culture, Sports, Science and Technology (MEXT). This work was performed in part at the Program of the Network‐Type Joint Usage/Research Center for Radiation Disaster Medical Science at Hiroshima University, Nagasaki University, and Fukushima Medical University.

CONFLICT OF INTEREST STATEMENT

YM received the research funds from Amano Enzyme Foundation for Science and Technology and from DAIKO FOUNDATION. These funds have no relevance to the content of this article. Other authors have no conflict of interest.

ETHICS STATEMENT

Approval of the research protocol by an institutional review board: N/A.

Informed Consent: N/A.

Registry and the Registration No. of the study/trial: N/A.

Animal Studies: Protocols were approved by the Institutional Animal Care and Use Committee of Hiroshima University (permission no. A21‐27).

Supporting information

Figure S1.

Table S1.

Sasatani M, Xi Y, Daino K, et al. Rev1 overexpression accelerates N‐methyl‐N‐nitrosourea (MNU)‐induced thymic lymphoma by increasing mutagenesis. Cancer Sci. 2024;115:1808‐1819. doi: 10.1111/cas.16159