The gene PPM1D (protein phosphatase Mg2+/Mn2+-dependent 1D) encodes for a phosphatase that negatively regulates the DNA damage response (DDR) by dephosphorylating multiple DDR proteins, including p53. Truncating mutations in exon 6 of PPM1D are among the top somatic mutations in age-related clonal hematopoiesis (CH) and therapy-related myeloid neoplasms (t-MNs). We recently demonstrated that PPM1D-mutant hematopoietic cells have selective growth advantage over their wild-type (WT) counterparts after exposure to specific chemotherapeutic agents, such as cisplatin, due to decreased apoptosis. However, the relationship between the increased survival of PPM1D mutants and their DNA repair capacity, as well as how they contribute to the progression of CH to t-MNs, is unclear.
To determine how aging and genotoxic stress affect the DNA repair capacity of PPM1D mutants, and to model the evolution and outcomes of Ppm1d-mutant CH in mice.
We examined the effect of PPM1D truncating mutations on the DNA damage response and DNA repair capacity using PPM1D-mutant cell lines and our Ppm1dR451X mice. We performed competitive transplantation experiments to study the short- and long-term effects of genotoxic stress and aging on the clonal dynamics of Ppm1d mutants in recipient mice.
We first compared the DDR of PPM1D mutant and WT cells in the context of irradiation, cisplatin, and the absence of stress. We found that PPM1D mutations stabilized the PPM1D protein, which then constitutively dephosphorylated downstream DDR pathway members, including ɣ-H2AX, p53, and Chk2. To understand whether this DDR suppression impairs DNA damage repair, we exposed PPM1D mutant and WT cells to gamma irradiation and cisplatin, and monitored DNA repair via CRISPR-based assays and the comet assay. In both cell lines and primary hematopoietic cells, we saw no significant difference in comet tail moments between Ppm1d-mutant and WT cells in the absence of stressors. However, the mutants had significantly longer tail moments after genotoxic stress (i.e. p < 0.01 after 16 hours of cisplatin treatment), suggesting impaired short-term DNA break repair.
To explore the long-term outcome of Ppm1d CH, we performed competitive bone marrow transplantation at a 20:80 ratio of Ppm1d-mutant to WT cells, and monitored mutant chimerism over one year. In the absence of prior genotoxic exposure, Ppm1d-mutant chimerism remained at similar proportions. In contrast, we observed a significant selection for Ppm1d mutants in the peripheral blood of the recipient mice treated with 5 weekly cycles of cisplatin, as early as 2 weeks into treatment. Interestingly, these Ppm1d clones persisted long after the conclusion of cisplatin treatment but did not expand, suggesting that PPM1D mutants gained an initial fitness advantage after genotoxic stress that was later maintained. Of note, one year after initial transplant, no clear signs of hematologic malignancies were observed.
Consistent with our previously published data, we observed increased dephosphorylation of multiple critical DNA repair proteins by mutant PPM1D. Furthermore, PPM1D mutants appear to have impaired short-term DNA break repair, as observed in the context of genotoxic stress. The absence of frank hematologic malignancies thus far in mice suggests that PPM1D may not be a potent oncogenic driver, but could provide hematopoietic cells with a survival advantage that facilitates future acquisition of cooperating mutagenic hits. These findings shed insight on the role of PPM1D in the development of t-MNs.