In this study, we for the first time demonstrate that APOBEC3B is a critical factor that induces p53-mutation and drug resistance of DLBCL. P53 is an important key factor that regulating of cell proliferation, DNA repair, and apoptosis. TP53 somatic mutations were identified in many types of cancer and were regarded as an important carcinogenesis and drug-resistant mechanism in many cancers10. Recent studies identified that TP53 mutation rate is about 20%-30% in DLBCL, with a similar incidence in germinal center B-cell-like (GCB) and activated B-cell-like (ABC) subtypes7–9, 19. While in present study, 3D-PCR-based sequencing method could identify TP53 exon8 mutation rate at about 35.9% in whole cohort, which is higher than previous data. Even in non-R/R DLBCL cases the TP53 exon8 mutation rate is 23.26%. This maybe could be explained by selective amplification of DNA fragments containing G/C to A/T mutation20. These results suggest that 3D-PCR could detect the G/C to A/T mutation in a higher sensitivity than that of regular PCR method. But this method also has a shortcoming of missing of other mutation types. While as previous reported that G/C to A/T mutation type is the main mutation type in TP53 gene, and most of hotspot mutations were G/C to A/T mutation10, so 3D-PCR also could be used as a valuable method for TP53 mutation detection.
As an important tumor genetic factor, TP53 mutation was included as a poor prognostic factor for acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) in National Comprehensive Cancer Network (NCCN) guidelines as well as for multiple myeloma in Mayo Clinic sMART system. In recent years, several studies indicated that TP53 mutation is also a poor prognostic factor for DLBCL7–9. However, this relationship has not been consistent because of some controversial data that failed to demonstrate any correlation between TP53 mutations and prognosis21, 22. In present study, results showed that TP53 exon8 G/C to A/T mutation was higher in R/R DLBCL patients than that of non-R/R group. Most of those mutations were missense mutation and caused amino acid change. And the predictive effect of TP53 exon8 mutation was also studied. Results showed that TP53 mutation group had a lower CR rate compared with that of wild-type group when treated with standard R-CHOP. Then the prognostic value of TP53 mutation status was analyzed in different IPI groups, which is the most wildly used prognostic factors model. Results showed that TP53 hotspot mutation also could discern patients with significantly distinct outcomes in IPI > 2 group. On the other side, the group with high risk IPI but wildtype & non-hotspot TP53 had the similar outcome with those IPI 0–2 group. Also, multivariate Cox model showed that TP53 hotpot was the strongest prognostic factor for PFS among TP53 mutation, IPI, LDH, stage and gender. These data indicate that TP53 hotspot mutation status provide robust prognostic information that is not captured by IPI.
Similar with previous reports, our data also showed that TP53 mutation could be regarded as a prognostic factor for DLBCL. In present study, only exon8 was sequenced and analyzed, this may underestimate the proportion of TP53 mutations. TP53 mutations of other exons also need to be detected to perfect this relationship. But this does not prevent the role of TP53 mutations in the prognosis of DLBCL.
Among the TP53 missense mutations, about a third of these mutations are located in six “hotspot” residues that are p.R175, p.G245, p.R248, p.R273, and p.R28223. Most of the previous reports did not analyze the differential prognostic value of different TP53 mutation positions in DLBCL. Young KH et al. found that TP53 mutations in DNA binding domain (DBD), especially in LSH and L3 regions, were associated with worse survival8. Xu-Monette et al. reported that special codons including 248, 273, 175, 176, and 213 of the p53 protein had the highest mutation frequency and associated with poor prognosis9. These indicated that hotspot mutations maybe more important for p53 activity and were more valuable for prognostic prediction. In present study, we also found that some hotspot mutation rate, such as 273, 282 and 283, were even higher in R/R DLBCL than that of non-R/R DLBCL. Those non-hotspot mutation patients had the similar CR rate and PFS with wild type group. But this need to be further studied because the non-hotspot mutation group was small in present study. These hotspot mutations, instead of non-hotspot mutations, were strongly associated with lower CR rate and shorter PFS in DLBCL. Previously data showed that these hotspot mutations could affect the DNA binding activity of p53 protein and result in failure of regulating of target genes24–26. Some of these hotspot p53 mutants were responsible for gaining of function in carcinogenesis and drug resistance27–30. For example, R273C and R273H mutants were reported to confer a more aggressive phenotype on cancer cells, as well as enhance resistance to DNA damaging drugs31. More recently, Boettcher S also found that TP53 missense mutations, including R273H and R282W, showed a dominant-negative effect, rather than gain of function, and caused drug resistance in AML cells32. Our data found that the hotspot mutations instead of all the mutation sites of TP53 were associated with worse survival of DLBCL. Which hotspot mutations are more important for prognostic evaluation of DLBCL and how these hotspot mutations affect the role of p53 in tumorigenesis and drug resistance are still unclear.
TP53 mutation could result in resistance to chemotherapy agents such as doxorubicin32 and cisplatin33–36, which are usually used for DLBCL. Although there were some attempts for overcoming of TP53 mutation but this still needs further study. Lack of understanding of TP53 gene mutation mechanism is one of the important obstacles to overcome p53 mutant-mediated drug resistance. Among the reported TP53 mutation types, G/C to A/T is the most common type, especially some hotspot mutation sites. Most of the reported carcinogens, such as PAH (B[a]P), AA, aflatoxin B1, Vinyl chloride and 3-NBA, doesn’t induce G/C to A/T mutation10. Except for the UV radiation was reported could inducing CC to TT mutation in skin cancer37. But the mechanism of the majority G/C to A/T mutation in TP53 is still unclear. Previously our group and other groups had identified that some APOBEC3s family members could induce G/C to A/T mutation in viral genome including human immunodeficiency virus (HIV)38–40, human T-lymphocytic leukemia virus (HTLV-1)41–44, and hepatitis B virus (HBV)15,45−47. In recent years, APOBEC3B is reportedly one of the most extensive candidate factors for studying G/C to A/T mutations in a variety of human cancers14,48−50. Moreover, some reports proved that APOBEC3s could be up-regulated by interferon51,52, which is an important cytokine involved in inflammation. And chronic inflammation was regarded as a potential factor in lymphoma carcinogenesis. So, we raise the hypothesis that APOBEC3s, especially APOBEC3B, may be responsible for TP53 G/C to A/T mutation.
Among the seven family members (from APOBEC3A to APOBEC3H), APOBEC3B localized mainly in nucleus, APOBEC3G, APOBEC3F and APOBEC3DE localized mainly in cytoplasm, and APOBEC3A and APOBEC3C localized both sides15,47,53. Based on bio-informatics analysis of APOBEC3s expression in DLBCL, we found that APOBEC3B not APOBEC3A, was up-regulated in DLBCL in several lymphoma database. This suggests that APOBEC3B may be more possibly responsible for TP53 mutations in DLBCL.
Using inducible expression system, we over express APOBEC3B in DLBCL cells, and found that G/C to A/T mutation could be induced in TP53 exon8. More importantly, these in-vitro mutation patterns were as the same as that of in-vivo R/R DLBCL samples. Those hotspot mutants were also could be detected in APOBEC3B-induced p53 mutants as the similar pattern of that of DLBCL samples. While over expression of APOBEC3A could not induces G/C to A/T mutation in TP53. Also, c-MYC, which is less mutated in DLBCL54–56, was not mutated after APOBEC3B expression in DLBCL cells. These results indicated that APOBEC3B, instead of APOBEC3A, may responsible for the TP53 G/C to A/T mutation in DLBCL. APOBEC3B could selectively induce G/C to A/T mutation in special genes through an unknown mechanism in DLBCL. Further study found that these APOBEC3B-induced mutants could improve the proliferation of DLBCL cells as well as cause resistant to doxorubicin, one of the main drugs of CHOP. This drug resistance maybe could be explained by previous work by Li J31 and Boettcher S32. These results indicated that APOBEC3B-induced p53 mutants maybe responsible for the refractory and resistant of DLBCL. As APOBEC3B target different sequence in different cells, how APOBEC3B selective the targeting sequence and which factors were involved in this process needed to be further investigated. Recently, p53 protein was reported could regulate the expression of APOBEC3B 57, and APOBEC3B expression also increases APOBEC signature mutation in p53-defective cells58. So, the detailed crosstalk between p53 and APOBEC3B, during carcinogenesis and drug resistance, is still needed to be further investigated.