The Role Of E3 Ubiquitin Ligase WWP2 And The Regulation Mechanism Of PARP1 Ubiquitination In Acute Lymphoblastic Leukemia

Objectives:Acute Lymphoblastic Leukemia(ALL)is a disease caused by uncontrolled proliferation and malignant transformation of hematopoietic stem cells,which eventually invade the bone marrow and blood.With the development of medical technology,the 5-year disease-free survival rate of children with acute lymphoblastic leukemia can reach more than 90%,but the current treatment of ALL still has limitations.So far,chemoradiotherapy as the mainstream treatment of ALL is still limited by myelosuppression,cardiac toxicity and other toxic side effects.Although bone marrow transplantation has been incorporated into the treatment options to help rebuild the blood system and immune system damaged by intensive chemotherapy and radiotherapy,the prognosis of patients with relapsed and refractory ALL remains poor.In adults,the 5-year disease-free survival rate of ALL patients is only 35-45%,and most adult ALL patients,including some pediatric ALL patients,are facing the risk of relapse and refractory.Therefore,it is of great clinical significance to explore the molecular mechanism of ALL and find new molecular markers as well as effective therapeutic targets for ALL.Post-Translational Modification(PTM)is one of the most important protein regulatory mechanisms,including ubiquitination,phosphorylation,acetylation,methylation,glycosylation and SUMOylation.Among them,ubiquitination is the process by which ubiquitin molecules are linked to substrate proteins or themselves in a covalent form through its C terminus.According to the number of linked ubiquitin molecules,ubiquitination can be divided into mono-ubiquitination and polyubiquitination.Mono-ubiquitination often changes the interaction,localization and transport of protein substrates,while polyubiquitination is mainly related to proteasome-dependent substrate degradation,activity and translocation.The ubiquitin-proteasome system(UPS)is a large and complex protein network and plays an important part in occurrence and development of ALL,which is composed of ubiquitin activating enzymes(E1),ubiquitin conjugating enzymes(E2)and ubiquitin ligase enzymes(E3).Among them,E3 ubiquitin ligases are particularly important in the UPS due to their structural properties to specifically recognize substrates.WWP2(WW domain-containing protein 2)is one of the main members of NEDD4family and belongs to the HECT type E3 ubiquitin ligase.WWP2 is widely expressed in human heart,placenta,lung,liver,muscle,kidney,pancreas and brain.The transcription level is highest in skeletal muscle,peripheral leukocytes,placenta,heart and kidney,higher in brain,spleen and lung and lower in the thymus,liver,colon,and intestine.Recent studies have shown that E3 ubiquitin ligase WWP2 participates in and regulates many biological processes such as cell cycle,immune response,apoptosis and cell signal transduction through ubiquitination modification of different substrates,and also participates in the regulation of the proliferation of malignant tumors such as liver cancer,lung cancer and gastric cancer.PARP1(Poly ADP-ribose Polymerase 1)is the principal member of the PARP family.At present,PARP1 has been extensively studied and is best known to play a protective role in a variety of DNA damage repair processes.However,other studies have shown that the excessive activation of PARP1 caused by oxidative stress can lead to excessive consumption of intracellular ATP and NAD+and release of ROS in mitochondria,thereby promoting cell apoptosis and death.In addition,PARP1 can coordinate the structural changes of chromatin and also play a role in chromatin remodeling.Because of key roles of PARP1 in a variety of cellular activities,it has been considered as a potential therapeutic target for several malignant tumors.This study aims to explore the role and mechanism of WWP2 in ALL,and to provide new evidence and new direction for finding potential therapeutic targets for ALL.Methods:1.Investigate the difference of WWP2 expression in bone marrow blood between normal controls and ALL patients at different stages.The bone marrow blood samples of normal control population,newly diagnosed ALL patients,complete remission ALL patients and relapsed ALL patients were collected for extracting mononuclear cells.After RNA extracting,genomic DNA removing and RNA reverse transcription,the relative expression difference of WWP2 was verified by RT-q PCR.2.Investigate the association between WWP2 expression level and clinicopathological factors in newly diagnosed ALL patients.The clinicopathological information of ALL patients(gender,age,FAB subtype,T/B classification,proportion of immature cells,WBC,Hb,PLT,BCR/ABL,karyotype)and WWP2 expression levels were collected and statistically analyzed.3.Investigate the effect of WWP2 on the growth of ALL subcutaneous xenograft.Four-weeks old nude mice were randomly divided into two groups,and the normal control Jurkat cells and sh WWP2 Jurkat cells were subcutaneously injected into nude mice in order to construct the ALL xenograft model.When the tumor volume exceeded 80mm~3,normal saline and doxorubicin(Dox)solution(2mg/kg/d)were injected intraperitoneally into nude mice once every two days for 7 times in each group.The growth status of nude mice and tumors in NC+Nacl group,NC+Dox group,sh WWP2+Nacl group and sh WWP2+Dox group were observed regularly,the horizontal and vertical axes of tumors were measured,and the volume of tumors was calculated.At 20 days after inoculation with Jurkat cells,the nude mice were sacrificed,the tumors were separated,weighed,and photographed.The volume growth curve and weight statistical chart of the xenograft tumor in nude mice were drawn.4.Investigate the effect of WWP2 on Dox-induced apoptosis in ALL transplanted tumor tissues.Immunofluorescence and Western Blot were used to verify the knockout efficiency of WWP2 in ALL subcutaneous xenograft tumors and the success of the construction model.The protein solution was extracted from part of the tumor tissues,and the expression levels of apoptosis marker proteins(Bax and Cleaved-Caspase-3)were verified by Western blot.5.Investigate the regulation and interaction between WWP2 and PARP1 in ALL xenograft tissues.Western Blot was used to verify the expression levels of WWP2 and PARP1 in NC+Nacl group,NC+Dox group,sh WWP2+Nacl group and sh WWP2+Dox group.Co-immunoprecipitation(co-IP)was used to verify the interaction between WWP2 and PARP1 in the NC+Nacl group,NC+Dox group,sh WWP2+Nacl group and sh WWP2+Dox group.6.Investigate the relationship between WWP2 and Dox-induced apoptosis in ALL cells.Gradient concentrations(0,0.05,0.1,0.1,0.2,0.4μM)of Dox were set to stimulate Jurkat cells,and the expression levels of WWP2 and apoptosis marker proteins(Bax and Cleaved-Caspase-3)were detected by Western Blot.CCK-8 assay was used to verify the cell viability stimulated by different concentrations of Dox.The concentration gradient(0,0.025,0.05,0.075,0.1μM)of Dox was set to stimulate Jurkat cells,and Annexin V-FITC/PI flow analysis was used to verify the apoptosis rate of cells stimulated by different concentrations of Dox.7.Investigate the effect of WWP2 overexpression on Dox-induced apoptosis in ALL cells.HA-control group,HA-control+Dox group,HA-WWP2 group and HA-WWP2+Dox group were set up by transfection of HA-control/WWP2 plasmid and Dox stimulation.The expression levels of WWP2,BAX,Cleaved-Caspase-3 and PARP1 were verified by Western Blot(0.2μM),cell viability was measured by CCK-8(0.2μM)and annexin V-FITC/PI flow cytometry analysis was used to verify the apoptosis rate in Jurkat cells under different treatments(0.075μM).Investigate the effect of WWP2 knockout on Dox-induced apoptosis in ALL cells.NC group,NC+Dox group,sh WWP2 group and sh WWP2+Dox group were set up by Dox stimulation.The expression levels of WWP2,BAX,Cleaved-Caspase-3 and PARP1 were verified by Western Blot(0.2μM),and cell viability was measured by CCK-8(0.2μM)and annexin V-FITC/PI flow cytometry analysis was used to verify the apoptosis rate of the cells under different treatments(0.075μM).8.Investigate the interaction and regulation between WWP2 and PARP1 in ALL cells.The interaction between PARP1 and WWP2 was verified by proteomic mass spectrometry.The interaction between WWP2 and PARP1 in Jurkat cells under normal conditions and Dox(0.2μM)stimulation was verified by co-IP.9.The expression levels and changes of WWP2 and PARP1 were verified by gradient overexpression and knockdown of WWP2.10.Investigate whether WWP2 negatively regulates PARP1 via proteasome pathway.Jurkat cells were treated with CHX(transcription inhibitor),MG132(proteosome inhibitor)or CQ(lysosome inhibitor)for 0h,3h and 6h,respectively.The half-life test was used to verify the expression level and change rate of PARP1 in the WWP2 overexpression and WWP2 knockout groups,respectively.Jurkat cells were treated with CHX,MG132 or CQ at the same time,and the normal control group,CHX group,CHX+MG132 group and CHX+CQ group were set up to verify the expression level of PARP1.11.Investigate whether WWP2 modifies PARP1 via polyubiquitination and mediates the ubiquitin-proteasomal degradation of PARP1.The interaction between WWP2 and PARP1was verified by co-IP under MG132 stimulation.Under the stimulation of MG132 and transfection of HA-Ub,WWP2 was overexpressed or WWP2 was knocked down to verify the ubiquitination level of PARP1 by co-IP.Results:1.The expression level of WWP2 in bone marrow blood of normal control population was different from that of ALL patients at different stages.The relative expression level of WWP2 in newly diagnosed ALL patients 0.0416(0.0219,0.2443)and relapsed ALL patients 0.0352(0.0109,0.0804)was higher than that in normal control group 0.0044(0.0022,0.0196).The relative expression level of WWP2 in newly diagnosed ALL patients 0.0416(0.0219,0.2443)was higher than that in complete remission ALL patients 0.0132(0.0036,0.0505).2.High WWP2 expression was associated with a high proportion of immature cells and a low PLT level in bone marrow blood of ALL patients.WWP2 expression level was not correlated with other clinicopathological factors such as gender,age,FAB subtype,T/B subtype,WBC level,Hb level,BCR/ABL external fusion gene and karyotype classification.3.WWP2 knockout inhibited the growth of ALL subcutaneous xenograft.4.Knockout of WWP2 enhanced Dox-induced apoptosis in ALL xenograft tumors.5.WWP2 negatively regulated PARP1 expression and interacted with PARP1 in ALL xenografts.6.WWP2 was associated with Dox-induced apoptosis in ALL cells.7.WWP2 overexpression down-regulated PARP1 expression and alleviated Dox-induced apoptosis and in ALL cells.WWP2 knockout up-regulated PARP1 expression enhanced Dox-induced apoptosis and in ALL cells.8.The results of proteomics mass spectrometry showed that PARP1 was one of the interaction proteins of WWP2.WWP2 interacted with PARP1 in Jurkat cells and was attenuated by Dox stimulation.9.WWP2 negatively regulated PARP1 expression.10.WWP2 negatively regulated PARP1 through the proteasome pathway.11.WWP2 polyubiquitinated PARP1 and mediated PARP1 degradation by ubiquitin-proteasome pathway.Conclusion:1.WWP2 is related to the progress of ALL;2.WWP2 promotes the growth of subcutaneous xenograft of ALL.3.WWP2 negatively regulates PARP1 by mediating ubiquitin-proteasomal degradation of PARP1 through polyubiquitination and alleviates apoptosis in ALL.