The Mechanism Of SUMOylation Regulates Alternative Lengthening Of Telomeres(ALT)

Alternative Lengthening of Telomeres(ALT)is a type of telomere maintenance mechanisms dependent on homologous recombination(HR)in about 10%-15% of hunman cancers.In ALT+ cancer cells,PML bodies can colocalize with telomeres,and form specialized structures called ALT-associated PML bodies(APBs).ALT telomeres DNA synthesis in APBs induces replication stress which driven SUMO-mediated feedback loop to promote self-perpetuating process.However,to maintain the balance of ALT activities,there must be a braking mechanism that prevents excessive activation of ALT.But,the regulatory mechanism remains unknow.Therefore,the study on the mechanism of ALT pathway is not only helpful to guide the development of drugs targeting ALT+ tumors,but expected to provide new theoretical support for the combination of telomerase inhibitor targeting tumors.Sumoylation is mainly regulated by SENP(Sentrin-specific Protease)family proteins and STUb L(Sumo-targeted Ubiquitin Ligases)proteins in cell.SENP6 proteins can mediate de SUMOylation of poly-SUMOylated PML protein.RNF4,a member of STUb L family,can mediaie the ubiquitination of poly-SUMOylation PML and induce their degradation.However,it is unclear whether SENP6 and RNF4 regulate the balance of ALT pathway by affecting the SUMOylation of PML bodies and other proteins on APBs.In this study,we use ATSA(ALT telomere synthesis in APBs)and found that SENP6 knockdown can significantly increase the number of APBs and the DNA synthesis of ALT telomeres in U2 OS cells(ALT+),suggesting that SENP6 is involved in the inhibition of ALT pathway.Then,employing Immunofluorescence(IF)and Fluorescence in situ hybridization(FISH)techniques,we found that SNEP6 could not only specifically locate APBs sites and SENP6 knockdown resulted in significant accumulation of SUMOylation at APBs.These results suggest that SENP6 may mediate the negative feedback loop to inhibit ALT pathway activity.Our further study shows that RNF4 is also involved in inhibiting ALT signaling.According to IF-FISH,RNF4 knockdown can significantly increased the number of APBs and ALT telomeres DNA synthesis,and RNF4 directly binds to the APBs site and inhibits the accumulation of SUMO modification on ALT telomeres.Subsequently,we found that RNF4 knockdown resulted in significant enrichment of BLM,RAD52 and RPA proteins in ALT telomeres.Considering that RNF4 could binds SUMOylated proteins and mediate their ubiquitination an degradation,we speculated that RNF4 could bind APBs sites and inhibit the accumulation of SUMOylation on APBs through ubiquitination activity,thus negatively regulating the ALT pathway.According to the effects of different time of ATO treatment on ALT pathway,we found ATO may inhibit ALT pathway through two mechanisms: 1)Short-term(6 h)ATO treatment of U2 OS cells significantly induce the SUMOylation of PML and aggregation of PML bodies,and significantly inhibites APBs and telomeres DNA synthesis.Since PML did not degrade after ATO treated at 6h,we speculated that excessive SUMOylation of PML caused by ATO in the short-term inhibites the signal of ALT pathway,by destroying the dynamic balance of APBs itself.2)Long-term(48h)ATO treatment induced PML degradation,which inhibited APBs and telomere DNA synthesis signals more significantly,and further experiments show that ATO could also inhibit the accumulation of BLM,RAD52 and RPA signals at telomeres sites.Finally,knocking down RNF4 can partially reverse the inhibition effect of ATO long-term treatment on ALT pathway,suggesting that RNF4 participate the process of ATO inhibition ALT.In summary,SENP6 and RNF4 can localize at APBs,inhibite the accumulation of SUMO on APBs and prevent overactivation of ALT pathway.Therefore,SENP6 and RNF4 can mediate the negative feedback loop signal of ALT pathway to balance the activity of ALT in telomere maintenance.Furthermore,we explore that ATO can targeted PML and suppress formation of APBs to blocking ALT,which support novel theory to treat ALT+ tumors.