NOTCH1 INHIBITS THE DNA DAMAGE RESPONSE BY IMPAIRING THE FORMATION OF THE ATM-FOXO3A-KAT5 COMPLEX

The DNA damage response is a pathway responsible for the maintenance of genome integrity. In my thesis I focused on the investigation of the modulation of ATM activity, a DNA damage response master kinase, by NOTCH1 receptor. Here I show that NOTCH1 inhibits DNA damage response activation. This inhibitory effect of NOTCH1 is not mediated by its transcriptional activity, but it is the result of direct binding between NOTCH1 and ATM kinase. I show that NOTCH1 binds to the FATC domain of ATM, and this results in an inhibition of ATM kinase activity. Furthermore, I provide evidence that NOTCH1-mediated ATM inhibition does not result from the impairment of ATM recruitment to DNA double-strand breaks. Rather, I show that NOTCH1 competes with FOXO3a transcription factor for the binding to the FATC domain of ATM and that over-expression of FOXO3a prevents NOTCH1-mediated ATM inhibition. As the exact function of FOXO3a in ATM activation was unclear, I sought to understand molecular mechanisms underlying NOTCH1-mediated ATM inactivation and the role of FOXO3a as an opposing factor in this process. I discovered that FOXO3a forms a direct complex with KAT5 lysine acetyl transferase that is critical for ATM activation upon DNA damage. Moreover, I observed that FOXO3a was necessary for the formation of a complex between ATM and KAT5. Surprisingly, I observed that NOTCH1 was not only impairing ATM-KAT5 interaction, but also FOXO3a-KAT5 one. This unexpected observation led me to the discovery that FOXO3a-KAT5 interaction is restricted to the formation of this three-protein complex together with the ATM kinase. Next, I demonstrated that induction of FOXO3a nuclear localization as well as inhibition of NOTCH1 increases ATM activation in NOTCH1-driven cancer cells, which leads to augmented DNA damage-induced cell death. Finally, I show that, in addition to ATM, NOTCH1 interacts also with other PI3K-like kinases: DNA-PKcs and ATR. Although I did not observe a significant impact of NOTCH1 on ATR kinase activation in the experimental settings I used, I observed an impaired activation of DNA-PKcs, which however did not result in a significant reduction of DNA damage repair in NOTCH1-expressing cells.