Effects And Mechanism Study Of Ubiauitin-Proteasome-Dependent Degradation Of CDK2 On Inducing Differentiation Of Acute Myeloid Leukemia Cells

IntroductionSince the first application of ATRA for the treatment of APL several decades ago,differentiation therapy has been viewed as a promising and revolutionary approach for the treatment of AML.However,ATRA is considered to be the sole efficient agent for differentiation-based therapy for patients with leukemia,and only APL patients respond to ATRA treatment.Thus,the development of new and effective differentiation therapies is clearly required,and research strategies seeking to identify novel therapeutic targets based on mechanistic studies are urgently needed.Our previous study showed that during ATRA-induced granulocytic differentiation in AML cells,Cyclin-dependent kinases 2?CDK2?protein undergoes a marked decline.Moreover,the central role attributed to CDK2 in cell cycle progression has recently been challenged by the observation that mice lacking this kinase develop normally.Interestingly,several independent studies strongly support the significance of the reduction of CDK2 in the regulation of self-renewal capacity and normal cell development.This linkage and the nonessential role in cell cycle regulation have further led us to focus on the differentiation-blocking role of CDK2 in cancer cells,especially AML cells.Therefore,whether and how CDK2 might be degraded by UPP in human AML cells and the role of CDK2 degradation on AML cells differentiation are intriguing avenues of investigation,which has positive significance for identifying novel differentiation therapeutic strategies.ObjectiveThe aim of this study was to investigate the effects and mechanisms of ubiquitin-proteasome-dependent degradation of CDK2 on inducing differentiation of acute myeloid leukemia cells.The current study included three sections:1)To investigate whether and how CDK2 underwent UPP-dependent degradation during AML cells differentiation.2)To explore the differentiation-blocking role of CDK2 in AML cells via knockdown or pharmacological inhibition of CDK2.3)To gain insight into the mechanism of CDK2 knockdown induced differentiation via performing microarray,and to identify the direct target of CDK2 using LC/MS/MS analysis.Part 1 The ubiquitin-proteasome-dependent degradation of CDK2 during AML cells differentiation and the detail mechanism of degradationMethodsHuman AML cells were treated with four differentiation-induction chemicals,including ATRA,12-O-tetradecanoylphorbol 13-acetate?TPA?,dimethyl sulfoxide?DMSO?and dasatinib?Dasa?;Cell differentiation induction was determined by assessing CD11b expression;Primary patient blasts from bone marrow of patients were isolated using lymphocyte monocyte separation medium;Real-time PCR was used to detect the mRNA level of CDK2;The proteasome inhibitor and lysosomotropic autophagy inhibitors were used to determine whether the decrease in CDK2 protein was resulted from the activation of UPP;Immunoprecipitation was performed to detect the interaction between CDK2 and Ub;The preferential ubiquitination sites of CDK2 were determined by observing the stability of CDK2 when lysines were replaced with arginine;To identify the ubiquitin E3 ligase responsible for the ubiquitination and degradation of CDK2,yeast two-hybrid?Y2H?screening was performed;We manipulated the KLHL6 levels by overexpression and shRNA,and then determined the stability of CDK2 protein.Results1)CDK2 is specifically degraded during granulocytic differentiation of human AML cells.? A distinct decrease was observed in CDK2 during the differentiation process in all AML cell lines:Four differentiation-induction chemicals?ATRA,TPA,DMSO and Dasa?were used to induce the differentiation of different AML cell lines as assessed by CD11b expression,and the Western blot results showed that the protein level of CDK2 was declined in all differented AML cells;Mirroring the results in the cell lines,the expression of CDK2 protein rapidly turned over in response to ATRA in primary AML samples?Leu-1-3?,together with an up-regulation of differentiation marker proteins?PU.1 and STAT1?;In contrast,the CDK2 levels remained stable in differentiated osteosarcoma U20S cells.? The change of CDK2 protein level did not result from the regulation of mRNA.Four differentiation-induction agents induced the differentiation of AML cells,but the mRNA level of CDK2 was rarely influenced by differentiation-inducing agents;?The decrease in CDK2 protein was resulted from the activation of UPP.The ATRA-induced decrease in CDK2 was completely abolished by MG132 treatment;Similar to ATRA,the TPA-induced decrease in CDK2 was also strongly arrested by MG132;In contrast,no obvious accumulation of CDK2 was observed in AML cells treated with ATRA plus chloroquine?CQ?or 3-methyladenine?3-MA?,two well-known lysosomotropic autophagy inhibitors.2)The mechanism involved in the ubiquitin-proteasome-dependent degradation of CDK2 during AML cells differentiation.? CDK2 was a substrate for ubiquitination.COS-7 cells were co-transfected with HA-tagged CDK2 and His-tagged Ub expression plasmids,and the results observe from immunoprecipitation experiment showed that a smear of high-molecular-weight ubiquitinated products?polyubiquitinated HA-CDK2?was detected;In addition,a subst antial anount of polyubiquitinated HA-CDK2 was further accumulated following MG132 treatment;Ubk48r,but not Ubwt or Ubk63r,greatly reduced CDK2 ubiquitination;?Lys-129 and Lys-142 were the preferential ubiquitination sites of CDK2.9 potential ubiquitination sites?Lys-20,Lys-24,Lys-3 3,Lys-129,Lys-142,Lys-237,Lys-250,Lys-273 and Lys-287?of CDK2 were replaced by arginine separately,and then analysis of a single lysyl replacement revealed that the turnover of K129R and K142R mutants was clearly impaired but not completely blocked;CDK2 with the replacement of two of the nine lysines?middle domain,M-2R?,the turnover defect was substantially augmented compared with any single mutants or other two domain mutants?N-3R,C-4R?;Using an "adding-back" approach,we further constructed a mutant CDK2 in which all nine lysines were replaced with arginine?9R?,each lysine was added back individually to 9R,among these 9 mutants,129K and 142K were consistently able to sustain the degradation of CDK2;?KLHL6 is the ubiquitin E3 ligase that mediates the degradation of CDK2.To identify the ubiquitin E3 ligase responsible for the ubiquitination and degradation of CDK2,yeast two-hybrid?Y2H?screening was used,and positive colonies were selected and found to harbor 10 prey vectors separately expressing TRIM10,ASB7,KCTD13,ZBTB20,PCGF2,RNF12,KLHL6,FBX032,FBXW2 and RNF148;The in vivo ubiquitination assay results illustrated that only Kelch like family member 6?KLHL6?promoted the accumulation of polyubiquitinated CDK2;Furthermore,the KLHL6-CDK2 interaction was confirmed by coimmunoprecipitation?CO-IP?;Overexpression of KLHL6 enhanced the degradation of CDK2 protein,whereas the depletion of KLHL6 prolonged the half-life of endogenous CDK2 protein.Part 2 Study on the relationship between CDK2 UPP-dependent degradation and AML cell differentiationMethodsTo further explore the effect of CDK2 on AML cell differentiation,three shRNA plasmids targeting CDK2?shCDK2#1,#2 and#3?were designed and introduced into AML cells;The total cell number and viability were determined by trypan blue exclusion with manual counting in Burker chambers;The induction of cellular differentiation was determined by assessing CD11b expression,a nitro blue tetrazolium?NBT?reduction assay and morphological changes;We re-expressed CDK2?CM14??a shCDK2#2-resistant mutant carrying synonymous mutations in its targeting sites?in CDK2 knockdown AML cells;To test whether the differentiation caused by shCDK2 was dependent on the kinase activity of CDK2,we use CDK2-specific inhibitors to detect the AML cell differentiation;The percentage of apoptotic cells induced by CDK2 inhibitors were identified as a sub-GO peak by flow cytometry;We evaluated the anti-tumor activity of CDK2 knockdown in AML subcutaneous xenograft nude mouse models,after the tumor reached?300 mm3,the mice received multiple intratumoral injections of the shRNA-encoding lentivirus targeting CDK2,and the inhibitory effect on tumor growth was monitored for different days;We intravenously transplanted AML cells transduced with scramble or shCDK2#2 lentivirus on NOD-SCID mice,and further assessed the in vivo effect of shCDK2 in NOD-SCID engraftment;The proportion of hCD45+mCD45" cells in the peripheral blood was analysed dynamically after transplantation to accurately reflect the overall leukemic burden;Finally,the length of the survival time of NOD-SCID mice was monitored.Results1)The effect of CDK2 protein level decline on the AML cell differentiation.?The influence of shCDK2 on AML cells pr.oliferation.All three shRNA?shCDK2#1,#2 and#3?could significantly reduce CDK2 expression to different extents,then clearly inhibited cell proliferation without inducing cell death;? The depletion of CDK2 drove myeloid differentiation in human AML cells.CD11b expression increased following these shRNA treatments in a time-dependent manner,peaking at day 7?from 5.39 ± 1.59%in the scramble control group to 33.7 ± 0.18%,55.0 ± 6.09%and 40.7 ±2.54%in the shCDK2#1,#2 and#3 groups,respectively?;Similar differentiation effects of CDK2 knockdown were also observed using the NBT reduction assay;Wright-Giemsa staining results also showed that compared with scramble-transduced cells,CDK2-depleted cells had a mature morphology,with an increased cytoplasm-to-nuclear ratio and clear nuclear segmentation;shCDK1 and shCDK2 could inhibit cell proliferation in AML cells;CDllb expression levels and PU.l protein expression were up-regulated by CDK2 depletion in a time-dependent manner in AML cells,whereas CDK1 depletion had no effect on CDllb expression;?Depletion of CDK2 in clinical AML samples led to relief of the differentiation blockade.3 primary patient samples?Leu-1-3?collected from AML patients were also transduced with the shCDK2 lentivirus,all of the patient samples showed a general tendency towards an increase in the expression of maturation markers,including an up-regulation of CDllb expression,mature morphological changes,and induction of STAT1,PU.1,and CEBP? expression.2)The influence of CDK2 kinase activity inhibition on the AML cell differentiation.? Dominant-negative mutant CDK2-D145N regulated the AML cells differentiation process.Knocking down CDK2 was sufficient to induce AML cell differentiation;However,the effect of shCDK2 was clearly inhibited by re-expressing wild-type CDK2;In contrast,the expression of a dominant-negative mutant CDK2-D145N?CM 14?failed to rescue the cells from differentiation.?Pharmacological inhibitors of CDK2 had a differentiation effect on AML cells.SU9516 had litter effect on apoptosis in all of the tested cell lines,while treatment with serial concentrations of SU9516 for 72h resulted in increased expression of CDllb in all three AML cell lines;The results of the NBT reduction assay were also in agreement with our hypothesis that inhibition of CDK2 by SU9516 could induce the differentiation of AML cells;Similar results were also obtained using two other CDK2 inhibitors?CVT313 and Nu6102?.3)Knockdown of CDK2 displayed the anti-cancer activity in vivo by driving AML cells differentiation.? Depletion of CDK2 arrests tumor growth and induces differentiation in human AML xenograft models.The average volume of tumors injected with a control lentivirus increased continuously,reaching an average volume of 2539 ± 386 mm3 compared with 969 ± 188 mm3 for the tumors injected with shCDK2 lentivirus?p<0.01?at the end of the experiment;After confirming the knockdown efficiency of shCDK2 lentivirus,we observed that more than 55.1%of the xenograft-derived HL60 cells were CD11b-positive compared with the control group?27.7%,p<0.01?;Similar tumor suppression and differentiation induction effects were noted in an NB4 xenograft nude mouse model after shCDK2 lentivirus treatment;?Depletion of CDK2 prolongs the survival of NOD-SCID mice inoculated with AML cells.We intravenously transplanted HL60 cells transduced with scramble or shCDK2#2 lentivirus on NOD-SCID mice,in the scramble groups,the population of hCD45+mCD45-cells increased from 2.04 ± 0.92%on day 32 to 6.91 ± 3.47%and 24.9 ± 8.03%on day 38 and 40 after the transplantation;However,in mice transplanted with HL60-shCDK2 cells,the population of hCD45+mCD45-cells remained undetectable?1.08 ± 0.53%,1.71 ± 1.33%and 1.34 ± 1.16%on day 32,38 and 40,respectively?;Furthermore,the leukemic blasts in mouse peripheral blood were dramatically reduced after CDK2 knockdown compared with the scramble group using the blood smear assay;Finally,overall survival was significantly improved in NOD-SCID mice transplanted with HL60-shCDK2 cells when compared with mice transplanted with scramble cells.Part 3 Study on the mechanism of CDK2 knockdown induced AML cells differerntiationMethodsReal-time PCR results were used to test the mRNA level of differentiation related factors;To gain insight into the mechanism of CDK2 knockdown induced differentiation,we performed microarray analysis in AML cells transduced with shCDK2;These altered genes were visualized with TreeView and clustered with David functional annotation clustering analysis;We knocked down MAFB expression level with shRNA;We also investigated CDK2 and MAFB expression in the xenograft-derived AML cells by western blotting and immunofluorescence;Western blot was used to check the degradation of PML/RARa;RARE-luciferase assay was employed to detect the RARa transcriptional activation;We performed co-inmunoprecipitation using anti-HA-beads in HA-CDK2 over-expressing AML cells,followed by liquid chromatography-tandem mass spectrometry?LS/MS/MS?analysis,with the aim of identifying proteins that interact with CDK2;The production of intracellular H₂O₂ was measured using the oxidation-sensitive fluorescent dye carboxy-DCFDA;The interaction between CDK2 and PRDXs was confirmed by performing direct co-immunoprecipitation studies;We determined the effect of shCDK2 on the expression levels of PRDX2 via western blot;Human CDK2 and PRDX2 were cloned into pET28a vector containing a His6 tag sequence at the N-terminal region,and these proteins were expressed in the Escherichia coli strain BL21?DE3?and purified;PRDX peroxidase activity was studied using the standard Trx/Trx reductase?TrxR?/NADPH-coupled spectrophotometric assay as described;To validate that PRDX2 is the endogenous target for CDK2 depletion-induced differentiation,we knocked down the expression of PRDX2 in AML cells by shRNA and then detected the differentiation level by assessing CD11b expression,a nitro blue tetrazolium?NBT?reduction assay and morphological changes.Results1)Exploration of the downstream pathway of CDK2 degradation induced differentiation,and searching of the key factor regulating the CDK2-driven differentiation.? Knockdown of CDK2 has a profound activation effect on the translation of its downstream pathwav.We performed microarray analysis in AML cells transduced with shCDK2?#1,#2 and#3?for 2 days?early differentiation stage?or 5 days?late differentiation stage?,35?18 upregulated and 17 downregulated genes?and 499?473 upregulated and 26 downregulated genes?displayed significant changes at day 2 and day 5,respectively;? MAFB is an important downstream of CDK2 to mediate differentiation of AML cells.Among these 16 genes,the mRNA levels of MAFB increased most dramatically,as upregulated 151-,64-and 252-fold in shCDK2#1,#2,and#3 on day 5,respectively;Similar to what we observed at the mRNA level,the protein expression of MAFB was obviously enhanced in shCDK2-transduced AML cells in a time dependent manner;The expression levels of CDK2 were indeed inhibited by shCDK2-lentivirus in xenograft-derived AML cells,whereas MAFB expression levels were specifically induced in shCDK2 group when compared with control group,and immunofluorescence results further confirmed these changes;MAFB-depletion had no effect on CD11b expression,while CDK2-depletion could increase CD11b expression at both day 2 and day 5 as expected;Moreover,shMAFB significantly reduced the percentages of CD11b+ cells in CDK2-depleted cells;Similar observations were also made with the NBT reduction assay.2)Investigation on the direct target and detail mechanism of CDK2 during AML cells differentiation.? The effect of CDK2 knockdown on PML-RARa fusion protein expression and RAR activation.Unlike ATRA,shCDK2 neither affected the level of RAR? nor cleaved or degraded PML-RAR?;Moreover,the RARE-luciferase assay also showed that CDK2 knockdown failed to modulate the RAR? transcriptional activation;? H₂O₂ functions as an indispensible role on the CDK2-decline induced AML cells differentiation.H₂O₂ accumulation was clearly induced by shCDK2 in AML cells;Moreover,N-acetyl-cysteine?NAC?,a H₂O₂ scavenger,notably abrogated the differentiation of AML cells induced by shCDK2 following the inhibition of H₂O₂ accumulation as assessed by CD11b expression and NBT reduction activity;?CDK2 targets and activates PRDX2 to arrest AML cell differentiation.Liquid chromatography-tandem mass spectrometry?LS/MS/MS?results showed that sixty-eight proteins were identified to physically interact with CDK2 in all three AML cell lines,including two important peroxiredoxins?PRDXl and PRDX2?;Both PRDXs?PRDX1 and PRDX2?were expressed in AML cells,but only PRDX2 was pulled down with CDK2;The expression levels of PRDX2 was relatively stable in the shCDK2-induced differentiated AML cells;rCDK2 effectively increased the peroxidase activity of recombinant PRDX2;According to the CD11b expression,morphological alterations and NBT reduction,knockdown of PRDX2 also induced the differentiation of all three AML cell lines.ConclusionIn this study,we show that CDK2 is specifically degraded through the UPP upon therapeutic differentiation of AML cells,and we demonstrate that the E3 ligase Kelch like family member 6?KLHL6?is responsible for the down-regulation of CDK2.We provide evidence that the depletion of CDK2 overcomes the myeloid differentiation blockade of AML cells both in vitro and in vivo.We further demonstrate that the administration of CDK2 pharmacological inhibitors enables the myeloid differentiation of AML cells.The differentiation function of CDK2 inhibition might be associated with the accumulation of H₂O₂ by directly inhibiting PRDX2 activity and following the reactivation of differentiation pathway.Together,our findings offer new insights into the role of CDK2 in differentiation of AML cells and suggest that targeting CDK2 may be a novel differentiation therapeutic approach for AML leukemia.