The Mechanism And Physiological Significance Of TOP2 Poison Trigger The Ubiquitination And Degradation Of TOP2?

DNA topoisomerase ?(TOP2)is a kind of complex molecular machine that modulate the topology of DNA by transiently breaking double-strand DNA and then re-ligating the ends of broken DNA,during RNA transcription,DNA replication,and chromosome condensation/de-condensation.It is essential for cell growth and survival.For this feature of TOP2,a variety of TOP2 targeting anti-tumor drugs have been developed,such as teniposide(VM-26),etoposide(VP-16),doxorubicin(DOX),and so on.These drugs will inhibit the re-ligating activity of TOP2 and poison the enzyme by stabilizing and increasing TOP2-DNA covalent complexes(TOP2ccs)and are therefore known as TOP2 poison.The formation of TOP2 ccs is the primary mechanism of TOP2 poison to kill tumor cells.Previous reports have shown that the drug-stabilized TOP2 will be degraded by 26 S proteasome and subsequent the exposure of doublestrand breaks(DSBs),which if failure to appropriately repaired will lead to genomic instability and chromosome rearrangement.Therefore,patients treated with TOP2-based drugs are associated with a high incidence of secondary malignancies and cardiotoxicity.Human genome encode two isozymes of TOP2 enzymes called TOP2? and TOP2?,independently.Previous research shows that the cytotoxicity of TOP2 poison is founded to be TOP2?-depedent,while the DNA sequence rearrangements and DSBs of TOP2 poison appeared to be mediated primarily by TOP2?.This is mainly due to the different degradation patterns of TOP2? and TOP2?.TOP2? is preferentially degraded over TOP2?,therefore the TOP2?-DNA covalent complexes level is relatively lower,but the DSBs mediated by TOP2? degradation is more severe.However,the underlying mechanism of TOP2? degradation and its subsequent biological effects remain unclear.If the mechanism of its degradation can be clarified in detail,it may provide new insight to improve the therapeutic effect of these agents and reduce the side effects.In this study,by using immunoblotting,immunofluorescence,gene knockdown/knockout,gene point mutation,in vivo ubiquitination assay,neutral comet assay,and other biochemical and molecular biology related technologies,we found that TOP2 poison-induced TOP2? degradation was mediated by SCF?-Tr CP ubiquitin ligase.Specifically,DNA damage signal,triggered by teniposide(VM-26)treatment,activates ATM and coordinates CK1 to phosphorylate TOP2? on Ser1134 and Ser1130,respectively,in a canonical degron motif,to facilitate ?-Tr CP binding and subsequent degradation.This conclusion is supported by the following evidence:(1)MLN4924 treatment significantly prolong the half-life of TOP2?,reduce its ubiquitination level,and in physiological conditions,MLN4924 can increase the protein level of TOP2? in a dose-depedent manner.(2)CULLIN 1 or ?-Tr CP silencing extends TOP2? protein half-life upon VM-26 treatment.(3)TOP2? S1130 A and S1134 A mutants,have longer protein half-life and reduction in ?-Tr CP-TOP2? binding and TOP2? ubiquitination.(4)Inactivation of ATM or CK1 by small molecular inhibitors or genetic knockdown/knockout abrogated TOP2? degradation,moreover ATM and CK1 bind to TOP2? directly.Biologically,blockage of TOP2? degradation in combination of VM-26 treatment increased the level of TOP2 ccs and impaired DNA damage response and repair,leading to accelerated cell death via apoptosis.The evidence is following:(1)Inactivation of SCF?-Tr CP,the ATM phosphorylation and ?H2AX levels caused by VM-26 were significantly reduced.(2)MLN4924 treatment or ?-Tr CP1 depletion caused a significant increase of the comet tail moment compared with the treatment of VM-26 alone or with sg Ctrl.(3)MLN4924 or ?-Tr CP1 depletion sensitize cells to VM-26 and leading to an accelerated cell death via apoptosis.(4)Expression of ?-Tr CPresistant TOP2? mutants sensitizes cells to VM-26.Thus,it appears that TOP2? degradation is a cellular defensive mechanism to facilitate the exposure of DSBs to trigger DNA damage response and repair.Collectively,our findings reveal a new strategy to improve the efficacy of TOP2-targeting anti-tumor drugs by combination with small molecule inhibitors of TOP2? degradation.