Post-translational Modification Of UCK1 Kinase To The Molecular Mechanism Of 5’-AZA Resistance

Objective:Acute myelogenous leukemia(AML)is a malignant tumor of hematopoietic system,characterized by abnormal proliferation of primary and juvenile myeloid cells in bone marrow and peripheral blood.DNA methyltransferase inhibitor azazine(5’-AZA),as a novel antitumor drug,has a good therapeutic effect on AML patients.However,in the course of clinical treatment,there is resistance to the drug.Therefore,it is of great significance to explore and further clarify the mechanism of 5’-AZA resistance and to find new therapeutic targets.Studies have shown that uridine-Cytidine Kinase 1(UCK1)can activate 5’-AZA,and its protein level is significantly down-regulated in drug-resistant patients.The potential molecular mechanism of reduced UCK1 expression has not been reported.The purpose of this study was to explore the association and molecular mechanism between the abnormal expression of UCK1 and 5’-AZA resistance in acute myeloid leukemia.Methods:We first constructed flag-labeled UCK1 plasmid,enriched target protein UCK1 by tandem affinity purification technology,and obtained proteins that could bind to UCK1 through mass spectrometry analysis.According to the protein binding score and literature,the potential binding protein KLHL2 was selected.Then,using molecular biology experiments,co-IP,GST-pulldown and fluorescence co-location to further confirm that KLHL2 and UCK1 can directly bind to each other,and identify the binding region of the two.Subsequently,qRT-PCR and Western blot were used to explore the association between the two,and in vitro and in vivo ubiquitination experiments were used to screen and identify the relevant ubiquitination sites.siRNA of UCK1 and KLHL2 or overexpression plasmid of UCK1 and KLHL2(HA-UCK1/Flag-KLHL2)were transfected into highly malignant AML cell lines respectively.CCK8 assay and flow cytometry were used to detect the effects of siRNA on proliferation,growth and apoptosis of 5’-AZA-treated cells.To clarify the biological function of KLHL2 to 5’-AZA.UCK1 fusion protein UCK1-LUC was constructed,and the deubiquitination enzyme USP28 that could stabilize UCK1 was screened by luciferase reporting experiment combined with the protein spectrum data in the early stage.Mutants and shRNA of USP28 were constructed,and the protein level of UCK1 was detected after cell lines were treated with actinomycin.To clarify the function of USP28 on UCK1 and the biological correlation between USP28/UCK1/KLHL2.By knocking down or overexpressing USP28 and UCK1 in the 5’-AZA-resistant strain R-HL-60,the significance of activation of ATM signaling pathway to USP28 and UCK1 was determined,and the phosphorylation sites of UCK1 were identified.The function of phosphorylated UCK1 and USP28 on 5’-AZA-treated cell lines was observed by adjacent junction technique(PLA).Finally,USP28 was overexpressed or knocked down in HL-60 cells,and cells were treated with different concentrations of 5’-AZA to detect cell proliferation,growth and apoptosis.At the same time,HL-60 cells transfected with or without USP28 plasmid were injected into acute myeloid leukemia mice,and then treated with 5’-AZA.The survival time of the mice was observed and recorded.Results:LC-MS showed that KLHL2 may interact with UCK1 protein.Co-IP and GST-Pull down experiments proved that UCK1 and KLHL2 could interact directly.By constructing truncated bodies of UCK1 and KLHL2,the GST-pull down experiment results showed that KLHL2 mainly interacts with the region 23-230 of UCK1.The ubiquitin assay demonstrated that KLHL2 was an E3 ligase of UCK1 and could ubiquitinate and degrade UCK1.The deubiquitinati on assay showed that USP28 was the deubiquitination enzyme of UCK1 and could stabilize the protein level of UCK1.According to the interaction region of UCK1 and KLHL2,we mutated the ubiquitination sites in this region one by one,the results showed that the binding of UCK1 to THE E3 ligase KLHL2 led to the ubiquitination and degradation of UCK1 at the K81 site.The deubiquitination enzyme(DUB)USP28 binds to UCK1 by interacting with KLHL2 and antagonizes KLHL2-mediated UCK1 ubiquitination.ATM substrate phosphorylation antibodies were used to demonstrate that USP28 and UCK1 could be phosphorylated by ATM.The deubiquitination of UCK1 by USP28 depended on the phosphorylation state of USP28.USP28 can be phosphorylated by ATM activated by 5’-AZA,leading to the separation of KLHL2 from USP28 and the instability of UCK1.ATM also phosphorylated UCK1 at S145 and significantly enhanced the formation of KLHL2-UCK1 complex.Down-regulation of KLHL2 gene not only significantly inhibited the proliferation of AML cells,but also sensitized AML cells to 5’-AZA and induced apoptosis.The combination of 5’-AZA and USP28 enhanced the inhibitory effect of 5’-AZA on AML cells and induced cooperative death in AML mouse models.Conclusion:Our current study shows that degradation of UCK1 caused by resistance to 5’-AZA is mainly realized by the ubiquitin-proteasome regulatory axis composed of KLHL2/USP28.Abnormal activation of ATM also plays an important role in drug resistance of 5’-AZA.On the one hand,ATM can phosphorylate the deubiquitination enzyme USP28,which successfully dissociates USP28 from UCK1 although it fails to change its enzyme activity,thus indirectly disrupting the stability of UCK1 protein.On the other hand,UCK1 is also the phosphorylated substrate of ATM,and the phosphorylated UCK1 increases its binding with E3 ubiquitin ligase,which directly leads to ubiquitin degradation of UCK1.Taken together,these findings suggest that the abnormal expression of USP28 and KLHL2 may have clinical reference and scientific value for evaluating the use of 5’-AZA.