PPM1D activity promotes the replication stress caused by cyclin E1 overexpression.

Oncogene-induced replication stress has been recognized as a major cause of genome instability in cancer cells. Increased expression of cyclin E1 caused by amplification of the CCNE1 gene is a common cause of replication stress in various cancers. Protein phosphatase magnesium-dependent 1 delta (PPM1D) is a negative regulator of p53 and has been implicated in termination of the cell cycle checkpoint. Amplification of the PPM1D gene or frameshift mutations in its final exon promote tumorigenesis. Here, we show that PPM1D activity further increases the replication stress caused by overexpression of cyclin E1. In particular, we demonstrate that cells expressing a truncated mutant of PPM1D progress faster from G1 to S phase and fail to complete licensing of the replication origins. In addition, we show that transcription-replication collisions and replication fork slowing caused by CCNE1 overexpression are exaggerated in cells expressing the truncated PPM1D. Finally, replication speed and accumulation of focal DNA copy number alterations caused by induction of CCNE1 expression was rescued by pharmacological inhibition of PPM1D. We propose that increased activity of PPM1D suppresses the checkpoint function of p53 and thus promotes genome instability in cells expressing the CCNE1 oncogene.

Truncated PPM1D Prevents Apoptosis in the Murine Thymus and Promotes Ionizing Radiation-Induced Lymphoma.

Genome integrity is protected by the cell-cycle checkpoints that prevent cell proliferation in the presence of DNA damage and allow time for DNA repair. The transient checkpoint arrest together with cellular senescence represent an intrinsic barrier to tumorigenesis. Tumor suppressor p53 is an integral part of the checkpoints and its inactivating mutations promote cancer growth. Protein phosphatase magnesium-dependent 1 (PPM1D) is a negative regulator of p53. Although its loss impairs recovery from the G2 checkpoint and promotes induction of senescence, amplification of the PPM1D locus or gain-of-function truncating mutations of PPM1D occur in various cancers. Here we used a transgenic mouse model carrying a truncating mutation in exon 6 of PPM1D (Ppm1dT). As with human cell lines, we found that the truncated PPM1D was present at high levels in the mouse thymus. Truncated PPM1D did not affect differentiation of T-cells in the thymus but it impaired their response to ionizing radiation (IR). Thymocytes in Ppm1dT/+ mice did not arrest in the checkpoint and continued to proliferate despite the presence of DNA damage. In addition, we observed a decreased level of apoptosis in the thymi of Ppm1dT/+ mice. Moreover, the frequency of the IR-induced T-cell lymphomas increased in Ppm1dT/+Trp53+/- mice resulting in decreased survival. We conclude that truncated PPM1D partially suppresses the p53 pathway in the mouse thymus and potentiates tumor formation under the condition of a partial loss of p53 function.

Radiation-induced synthetic lethality: combination of poly(ADP-ribose) polymerase and RAD51 inhibitors to sensitize cells to proton irradiation.

Although improvements in radiation therapy were made over the years, radioresistance is still a major challenge. Cancer cells are often deficient for DNA repair response, a feature that is currently exploited as a new anti-cancer strategy. In this context, combination of inhibitors targeting complementary pathways is of interest to sensitize cells to radiation. In this work, we used PARP (Olaparib) and RAD51 (B02) inhibitors to radiosensitize cancer cells to proton and X-ray radiation. More particularly, Olaparib and B02 were used at concentration leading to limited cytotoxic (alone or in combination) but increasing cell death when the cells were irradiated. We showed that, although at limited concentration, Olaparib and B02 were able to radiosensitize different cancer cell lines, i.e. lung and pancreatic cancer cells. Antagonistic, additive or synergistic effects were observed and correlated to cell proliferation rate. The inhibitors enhanced persistent DNA damage, delayed apoptosis, prolonged cell cycle arrest and senescence upon irradiation. These results demonstrated that radiation-induced synthetic lethality might widen the therapeutic window, hence extending the use of PARP inhibitors to patients without BRCAness.