MLN4924 suppresses the BRCA1 complex and synergizes with PARP inhibition in NSCLC cells.

Like ubiquitination, several studies have demonstrated that neddylation is implicated to be involved in the double strand break repair. BRCA1 is one of the key repair factors in the homologous recombination repair and may play a downstream role of the neddylation. BRCA1 is also a frequently mutated gene in cancers, which serve as the targets for PARP inhibitors. Here we further investigated the correlation between neddylation and BRCA1 complex using neddylation inhibitor MLN4924. MLN4924 efficiently inhibited the recruitment of components of BRCA1 complex to DNA damage sites. Thus MLN4924 may collaborate with PARP inhibitor to suppress tumor. Our results showed that combination MLN4924 and PARP inhibitor Olaparib impaired the DNA repair process in NSCLC cells. Furthermore, MLN4924 and Olaparib significantly inhibited the cancer cell growth. Kaplan-Meier survival analysis from lung cancer patients showed that high expression of NEDD8, BRCA1 and PARPs correlate with worse overall survival. Thus the combination of MLN4924 and PARP inhibitor may serve as a new strategy for NSCLC treatment.

TIP60 K430 SUMOylation attenuates its interaction with DNA-PKcs in S-phase cells: Facilitating homologous recombination and emerging target for cancer therapy.

Nonhomologous end joining (NHEJ) and homologous recombination (HR) are major repair pathways of DNA double-strand breaks (DSBs). The pathway choice of HR and NHEJ is tightly regulated in cellular response to DNA damage. Here, we demonstrate that the interaction of TIP60 with DNA-PKcs is attenuated specifically in S phase, which facilitates HR pathway activation. SUMO2 modification of TIP60 K430 mediated by PISA4 E3 ligase blocks its interaction with DNA-PKcs, whereas TIP60 K430R mutation recovers its interaction with DNA-PKcs, which results in abnormally increased phosphorylation of DNA-PKcs S2056 in S phase and marked inhibition of HR efficiency, but barely affects NHEJ activity. TIP60 K430R mutant cancer cells are more sensitive to radiation and PARP inhibitors in cancer cell killing and tumor growth inhibition. Collectively, coordinated regulation of TIP60 and DNA-PKcs facilitates HR pathway choice in S-phase cells. TIP60 K430R mutant is a potential target of radiation and PARPi cancer therapy.

RBX1 prompts degradation of EXO1 to limit the homologous recombination pathway of DNA double-strand break repair in G1 phase.

End resection of DNA double-strand breaks (DSBs) to form 3' single-strand DNA (ssDNA) is critical to initiate the homologous recombination (HR) pathway of DSB repair. HR pathway is strictly limited in the G1-phase cells because of lack of homologous DNA as the templates. Exonuclease 1 (EXO1) is the key molecule responsible for 3' ssDNA formation of DSB end resection. We revealed that EXO1 is inactivated in G1-phase cells via ubiquitination-mediated degradation, resulting from an elevated expression level of RING-box protein 1 (RBX1) in G1 phase. The increased RBX1 significantly prompted the neddylation of Cullin1 and contributed to the G1 phase-specific degradation of EXO1. Knockdown of RBX1 remarkedly attenuated the degradation of EXO1 and increased the end resection and HR activity in γ-irradiated G1-phase cells, as demonstrated by the increased formation of RPA32, BrdU, and RAD51 foci. And EXO1 depletion mitigated DNA repair defects due to RBX1 reduction. Moreover, increased autophosphorylation of DNA-PKcs at S2056 was found to be responsible for the higher expression level of the RBX1 in the G1 phase. Inactivation of DNA-PKcs decreased RBX1 expression, and simultaneously increased EXO1 expression and DSB end resection in G1-phase cells. This study demonstrates a new mechanism for restraining the HR pathway of DNA DSB repair in G1 phase via RBX1-prompted inactivation of EXO1.