Cell cycle- and DNA repair pathway-specific effects of apoptosis on tumor suppression Steven S. Fostera, Saurav Dea, Linda K. Johnsonb, John H. J. Petrinia,1, and Travis H. Strackera,c,1 aMolecular Biology Program, Sloan-Kettering Institute, New York, NY 10021; bCenter of Comparative Medicine and Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021; and cOncology Programme, Institute for Research in Biomedicine, 08028 Barcelona, Spain Edited by James E. Haber, Brandeis University, Waltham, MA, and approved May 8, 2012 (received for review January 12, 2012) The DNA damage response comprises DNA repair, cell-cycle check- required for subsequent activation of ATM, an apical kinase in point control, and DNA damage-induced apoptosis that collectively the DDR (2). ATM and related kinases are estimated to modify promote genomic integrity and suppress tumorigenesis. Previ- more than 700 substrates in response to DNA damage on con- ously, we have shown that the Chk2 kinase functions indepen- sensus SQ/TQ sites (6, 7). Among these substrates are key regu- dently of the Mre11 complex (Mre11, Rad50, and Nbs1) and ATM lators of cell-cycle checkpoints, DNA repair factors, and proteins in apoptosis and suppresses tumorigenesis resulting from hypo- that regulate apoptosis and senescence, accounting for the broad morphic alleles of Mre11 or Nbs1. Based on this work, we have physiological impact of DDR functions (8). proposed that Chk2 limits the oncogenic potential of replication- Arguably one of the most important ATM substrates is the associated DNA damage. Here we further address the role of Chk2 tumor suppressor p53. p53 controls the G1/S cell-cycle check- and damage-induced apoptosis in suppressing the oncogenic po- point and is required for both apoptosis and senescence in various cellular contexts (9). Deficiency of p53 results in severe predis- tential of chromosome breaks. We show that loss of Chk2 or a position to lymphoma in the mouse (10, 11). Mice harboring a mutation in p53 (R172P), which selectively impairs its function in separation-of-function mutation in p53, p53R172P, in which its apoptosis, rescued the lethality of mice lacking Lig4, a ligase re- apoptotic function is abolished but its influence on the G1/S quired for nonhomologous end-joining (NHEJ) repair of DNA dou- − − − − − − checkpoint and the induction of senescence remain intact, have ble-strand breaks in G0/G1. In contrast to Lig4 / p53 / mice, Lig4 / −/− −/− R172P/R172P been established. The spectrum and kinetics of tumor de- Chk2 and Lig4 p53 mice were not prone to organ- velopment in p53R172P/R172P mice are markedly distinct from GENETICS specific, rapid tumorigenesis. Although the severe NHEJ deficiency −/− R172P/R172P −/− those of p53 mice (12). p53 mice have a late-onset of Lig4 was a less potent initiator of tumorigenesis in the tumor phenotype, exhibiting lymphomas and a variety of solid R172P/R172P −/− p53 and Chk2 backgrounds, where p53 cell-cycle func- tumors, suggesting that senescence and G1/S checkpoint func- tions are largely intact, even mild defects in the intra-S and G2/M tions delay tumorigenesis in the context of apoptotic deficiency. checkpoints caused by mutations in Nbs1 are sufficient to influence Consistent with this view, the p53R172P allele retards the de- malignancy in p53R172P/R172P mice. We conclude that the oncogenic velopment of lymphomas resulting from Myc overexpression potential of double-strand breaks resulting from NHEJ deficiency is compared with p53 heterozygosity (13). Together, these data in- highly restricted by nonapoptotic functions of p53, such as the G1/S dicate that the apoptotic functions of p53 are not sufficient for its checkpoint or senescence, suggesting that the particular facets of role as a tumor suppressor. the DNA damage response required for tumor suppression are dic- p53 functions are activated by DSBs induced in G1 cells, leading to the activation of apoptotic as well as nonapoptotic tated by the proliferative status of the tumor-initiating cell. − − − − responses. Lig4 / and Xrcc4 / mice, which lack key compo- chromosome instability | kinase signaling | tumor suppressor nents of the NHEJ machinery, exhibit perinatal lethality and are associated with high levels of DSBs in areas of the brain con- taining differentiated and largely nondividing cell populations ollowing the detection of DNA lesions, the DNA damage re- −/− (14, 15). p53 deficiency rescues the lethality of Lig4 and sponse (DDR) coordinates DNA repair, cell-cycle checkpoints, −/− −/− −/− −/− −/− F Xrcc4 mice, but Lig4 p53 and Xrcc4 p53 mice have and specialized programs, such as apoptosis and senescence, to rapid onset of pro– and pre–B-cell lymphomas as well as me- prevent genomic instability (1). DDR genes frequently are mutated dulloblastomas with complete penetrance (16, 17). These data in chromosome instability and cancer predisposition syndromes, highlighting the role of the DDR in suppressing malignancy (2). indicate that defective NHEJ-mediated repair of DSBs arising in Markers of DDR activation are observed readily in premalignant G1 cells, such as those induced during antigen-receptor gene lesions, leading to the hypothesis that the DDR is an inducible assembly and in differentiated CNS components, are highly on- barrier to tumorigenesis (3). Indices of DDR activation are re- cogenic when p53 functions are lost (4). duced as tumors evolve, suggesting that selection against aspects of Previously, we provided evidence that the Chk2 kinase sup- DNA damage signaling accompany tumor progression (1). pressed the oncogenic potential of DNA replication-associated DNA damage through crosses with mice harboring mutations in DNA double-strand breaks (DSBs) are physiologically im- ATLD1/ATLD1 ΔB/ΔB portant lesions, because they can be cytotoxic or cytostatic and Mre11 (Mre11 ) and Nbs1 (Nbs1 ) (18). This in- generate oncogenic translocations (2, 4). Two major pathways of terpretation was based on the fact that those Mre11 complex DSB repair are used in the cell: nonhomologous end-joining alleles primarily affect the DDR in the S and G2 phases of the (NHEJ) and homology-directed repair (HDR). The use of HDR cell cycle and exhibit marked tumor predisposition in the context is cell cycle dependent, regulated by the activity of cyclin-de- pendent kinases and, in some cases, by the cell cycle-dependent expression or stability of required factors (5). HDR preferen- Author contributions: S.S.F., J.H.J.P., and T.H.S. designed research; S.S.F., S.D., and T.H.S. tially uses the sister chromatid as a template and thus is sub- performed research; S.S.F., S.D., L.K.J., J.H.J.P., and T.H.S. analyzed data; and S.S.F., J.H.J.P., stantially less error-prone than NHEJ, which typically results in and T.H.S. wrote the paper. the deletion or addition of DNA sequences at the junction of The authors declare no conflict of interest. repair. Deficiencies in both pathways have been linked to chro- This article is a PNAS Direct Submission. mosomal instability, DNA damage sensitivity, and cancer pre- Freely available online through the PNAS open access option. disposition, reflecting the hazards posed by DSBs (1). 1To whom correspondence may be addressed. E-mail: [email protected] or travis. The DDR is initiated by the recognition of DSBs that is me- [email protected]. diated primarily by the Mre11 complex, consisting of Mre11, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Rad50, and Nbs1. DSB engagement by the Mre11 complex is 1073/pnas.1120476109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1120476109 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 of Chk2 deficiency (19, 20). In contrast, Chk2 deficiency did not of DNA damage associated with DNA replication (12). We Δ enhance tumorigenesis in PrkdcScid mice, in which DSBs induced combined p53R172P/R172P with two Nbs1 mutants, Nbs1 B and Δ in G1 cells during antigen-receptor gene assembly are not re- Nbs1 C, which impair the response to DNA damage in S phase. paired (18). These data support the view that Chk2 tumor-sup- The N terminus of Nbs1 contains a Forkhead-associated (FHA) pressive activity may be relevant primarily for DNA damage that and tandem Brca1 C-terminal (BRCT) phosphopeptide binding arises during S or G2 phase (18). motifs, and the C terminus of Nbs1 contains a conserved domain In this study, we examine genetic contexts in which defective that contributes to the physical interaction between the Mre11 Δ apoptotic responses resulting from either p53 or Chk2 mutation complex and ATM (2, 21, 22). The Nbs1 C mutant lacks the C- are juxtaposed with DDR mutations that increase DNA damage terminal domain and exhibits an intra–S-phase checkpoint de- Δ in G1, S, or G2/M phases of the cell cycle. G1 DNA damage is fect, as well as a defect in apoptosis, whereas the Nbs1 B mutant, − − − − − − − − enhanced in Lig4 / mice. Unlike Lig4 / p53 / mice, Lig4 / lacking the FHA and first BRCT domain, is defective in both the − − − − Chk2 / and Lig4 / p53R172P/R172P mice were not highly prone intra-S and G2/M checkpoints but is competent for apoptosis to the rapid development of lymphoma and medulloblastoma. (19, 23). Δ Δ Together, these outcomes suggest that the oncogenic potential of Relative to p53R172P/R172P alone, both p53R172P/R172P Nbs1 C/ C Δ Δ breaks generated by NHEJ deficiency is restricted by non- and p53R172P/R172P Nbs1 B/ B double mutants exhibited reduced apoptotic p53 functions such as senescence or the imposition of survival, a more rapid onset of tumorigenesis, and alterations in Δ Δ the G1/S checkpoint. In contrast, Nbs1 C and Nbs1 B mutations, tumor spectrum (Fig. 1 A–D and Tables S1–S7). These results which impair the intra–S-phase and G2/M checkpoints, en- reveal that the intra–S-phase checkpoint defects associated with Δ Δ hanced tumorigenesis in p53R172P/R172P mice.