The Mechanism And Significance Of BCR-ABL Stabilization By Deubiquitinase USP7 In Chronic Myeloid Leukemia

ObjectivesChronic myeloid leukemia(CML)is a malignant proliferative disease derived from pluripotent hematopoietic stem cells.Its incidence in China is about 1/100,000 and it accounts for about 20%of all the adult leukemia cases.From the perspective of traditional Chinese medicine(TCM),this disease belongs to the categories of "blood syndrome","accumulation" and "deficiency and fatigue".So in clinical therapeutic principle,it is often treated by cooling the heat,dispersing blood stasis,detoxifying and replenishing vital qi.However,the mechanism of TCM playing the dominant therapeutic role in CML has not been elaborated.With the further study of CML in molecular biology,many scholars believe that the fusion gene BCR-ABL is one of the key factors leading to the occurrence and development of CML and drug resistance,therefore BCR-ABL has been the ideal therapeutic target of CML.Therefore,it is one of the purposes of this study to achieve the effective combination of traditional Chinese medicine and molecular biology in the clinical treatment of CML and to clarify its anti-CML mechanism.It is well known that the BCR-ABL protein can be degraded through the ubiquitin-proteasome pathway(UPP).Protein ubiquitination is a dynamic process directed by several key enzymes,including ubiquitin-activating enzymes(Els),ubiquitin-conjugating enzymes(E2s),ubiquitin ligases(E3s)and deubiquitinases(DUBs).Under the stepwise action of El,E2 and E3,the target proteins can be covalently added with ubiquitin molecules,while Dubs can hydrolyze the ubiquitin molecules or prevent ubiquitination from target proteins.Some ubiquitination enzymes have been reported for BCR-ABL ubiquitination,but its DUBs are yet to know.Therefore,the aims of the present study are:(1)to find the DUBs that prevent BCR-ABL ubiquitination;(2)to explore the mechanism of action and the influence of the DUBs on CML;(3)to screen out TCM molecules with bio-safety that can play an anti-CML role by participating in the above mechanisms,so as to provide scientific reference for CML targeted treatment of TCM.Methods1.Leukemia cell lines were collected for the evaluation of the expression of BCR-ABL protein by Western Blot(WB).Cells were treated with the proteasome inhibitors MG132 and Bortezomib(BZ),followed by WB assays to measure BCR-ABL protein levels.2.The BCR-ABL plasmid and individual DUB plasmids were co-transfected into HEK293T cells,the protein expression level of BCR-ABL was detected by WB,and the potential DUBs were idenitified based on the changes of BCR-ABL protein.3.After treatment with cycloheximide(CHX),cell stability and the half-life of BCR-ABL were analyzed by WB.4.The interaction between USP7 and BCR-ABL was analyzed by the Co-IP/WB assays.5.The effects of USP7 on the ubiquitination levels of BCR-ABL were detected by the IP/WB assays.6.Cells were treated with P5091,a small molecule inhibitor of USP7,the changes of the BCR-ABL protein and its ubiquitination levels were detected by the WB/IP assays.7.Lentiviral of USP7 was prepared to infect CML cells to investigate the effects of USP7 on the expression of endogenous BCR-ABL protein,the proliferation and apoptosis of CML cells,and the changes of the mRNA levels of BCR-ABL detected by RT-PCR.8.siRNA was transfected into CML cells to investigate the effects on the expression of endogenous BCR-ABL protein,the proliferation and apoptosis of CML cells,and the changes in the mRNA level of BCR-ABL detected by RT-PCR.9.Artesunate(ART),a natural small molecule compound,was used to treat CML cells followed by the evaluation of the protein level of BCR-ABL.10.Flow cytometry was used to measure the effects of ART on the apoptosis of CML cells,and WB was used to detect the changes of BCR-ABL protein levels after the combined treatment with ART and Imatinib(IM).Results1.BCR-ABL(p210)expressed in chronic myeloid leukemia cell lines K562 and MEGO1 but not in non-CML cells.We also found that it was the proteasome inhibitors MG 132 and BZ but not the lysosomal inhibitor CHQ increased the expression levels of BCR-ABL,which indicated that BCR-ABL could be degraded through UPP.2.The BCR-ABL plasmid was co-transfected with different DUB plasmids into HEK293T cells for 36 hrs,followed by protein extraction and WB assays,the results showed BCR-ABL was dramatically increased by USP7 and some other DUBs,but USP7 raised BCR-ABL in a highest level.3.After 36 hrs infection with lentiviral USP7,the CML cell line K562 was further treated with CHX for 0 to 24 hrs before being collected for WB assays to measure BCR-ABL levels,the results showed that USP7 significantly prolonged the half-life of BCR-ABL protein.4.The co-IP/WB assays showed that USP7 and BCR-ABL interacted with each other.5.After co-transfection of a BCR-ABL plasmid,a HA-Ub plasmid and a Flag-USP7 plasmid into HEK293T cells for 36 hrs,the IP/WB assays showed that USP7 reduced the ubiquitination levels of BCR-ABL and stabilized the BCR-ABL protein level in a time-and concentration-dependent manner.6.After being treated with P5091 for 12 hrs,K562 cells were collected for the IP/WB assays.The results showed that P5091 treatment markedly increased the ubiquitination levels of BCR-ABL,further suggesting that USP7 can modulate BCR-ABL ubiquitination and stability.7.After being infected with lentiviral USP7,K562 and MEG01 were subjected to WB and MTT assays.The results showed that overexpression of USP7 significantly up-regulated the endogenous BCR-ABL protein level,but had no effects on the mRNA levels of BCR-ABL.Consistently,USP7 promoted the proliferation of CML cells.8.After USP7 was knocked down from CML cell lines K562 and MEG01,the WB assay showed that the BCR-ABL protein but not its mRNA was consistently down-regulated.Accordingly,CML cell proliferation was also decreased.9.After treatment with artesunate(ART)for 24 hrs,CML cells were found to induce apoptosis because the annexin V positive cells were increased.Moreover,ART decreased BCR-ABL protein level in a concentration dependent manner and induce CML cell apoptosis.10.ART inhibited the interaction between USP7 and BCR-ABL and the combination of ART and Imatinib(IM)significantly down-regulated the protein level of BCR-ABL.ConclusionsThe above studies found that there exists a strong interaction between USP7 and BCR-ABL,and USP7 dramatically down-regulated the poly-ubiquitination levels of the BCR-ABL protein,therefore stabilizing BCR-ABL protein and increasing its half-life.The natural small-molecule compound ART disrupts the interaction between USP7 and BCR-ABL therefore reducing the protein level of BCR-ABL,which resulting CML cell apoptosis.All the present findings thus collectively demonstrate that USP7 is DUB of BCR-ABL,and USP7 could be a potential target for CML treatment.