DNA damage-induced phosphorylation of CtIP at a conserved ATM/ATR site promotes lymphomagenesis in NHEJ/p53-double deficient mice

Human lymphoid malignancies are often characterized by oncogenic translocations involving the antigen receptor gene loci. CtIP is a DNA end-resection factor that has been widely implicated in alternative end-joining (A-EJ) mediated chromosomal translocations in reporters. The ATM and ATR kinases phosphorylate CtIP at T859 (T855 in mouse) and other sites to promote DNA end-resection. Using two classical non-homologous end-joining (cNHEJ) and Tp53-double deficient mouse models, we identified a role of CtIP T855 phosphorylation in the neonatal development of Xrcc4-/-Tp53-/- mice and the IgH-Myc translocation driven lymphomagenesis in DNA-PKcs-/-Tp53-/- mice. Mechanistically, we found that CtIP T855 phosphorylation is important for the progression of DNA end-resection, while dispensable for hairpin opening and inter-sister DNA break ligation. Moreover, we found that CtIP-T855 phosphorylation supports the proliferation of Myc-driven lymphoma cells by promoting the transition from ATM-mediated to ATR-mediated G2/M cell cycle checkpoint. Correspondingly, the CtIP-T855A mutation delays splenomegaly in l-Myc mice. Collectively, our findings suggest that DNA damage-induced CtIP phosphorylation has a checkpoint function during lymphomagenesis independent of its role in A-EJ-mediated chromosomal translocation In this study, we examine how CtIP-mediated end resection contributes to A-EJ-mediated chromosomal translocations and lymphomagenesis by characterizing cNHEJ/Tp53-double deficient mice with or without the CtIP-T855A mutation. Specifically, we found that the CtIP-T855A mutation causes neonatal lethality in Xrcc4-/-Tp53-/- mice without apparent lymphomas or hematopoietic failure. Instead, the CtIP-T855A mutation exacerbate the G2/M arrest of Xrcc4-/-Tp53-/- olfactory neurons. In contrast, CtIPT855A/T855AKu70-/- mice are viable although small. Moreover, the CtIP-T855A mutation delays lymphomagenesis and alters the tumor spectrum of DNA-PKcs-/-Tp53-/- mice. High-throughput genome-wide translocation sequencing (HTGTS) of RAG- and endonuclease-initiated DSBs shows that CtIP T855 phosphorylation is not required for hairpin opening or the initiation of end-resection, but instead promotes extensive end-resection. Furthermore, CtIPT855A/T855A cells show defects in G2/M checkpoints maintenance, suggesting a role for T855 phosphorylation in lymphomagenesis beyond translocation. Correspondingly, the Ctip-T855A mutation also attenuates splenomegaly in the λ-Myc transgenic model. CJ and PPoI refer to the types of breaks or junctions that each sample was analyzed for. More specifically, CJ stands for "coding joins" which means that those samples specifically looked at the Coding Join junctions that formed during V(D)J recombination. Samples annotated with "PPoI" were analyzed for junctions formed at PPoI-endonuclease recognition cutting sites after the genome was exposed to the PPoI-endonuclease through retroviral infection and subsequent nuclear localization