CDK2‑instigates C/EBPα degradation through SKP2 in Acute myeloid leukemia
Gatha Thacker1 · Mukul Mishra1 · Akshay Sharma1 · Anil Kumar Singh1 · Sabyasachi Sanyal2,3 · Arun Kumar Trivedi1,2

Received: 20 March 2021 / Accepted: 10 May 2021 / Published online: 17 May 2021
© Springer Science+Business Media, LLC, part of Springer Nature 2021

Abstract
Transcription factor CCAAT/enhancer-binding protein-alpha (C/EBPα) regulates myelopoiesis by coupling growth arrest with differentiation of myeloid progenitors. Mutations in one or both alleles are observed in 10–14% AML cases that render C/EBPα functionally inactive. Besides, antagonistic protein–protein interactions also impair C/EBPα expression and function. In recent independent studies, we showed that CDK2 and SKP2 downregulated C/EBPα expression in an ubiquitin-dependent proteasome degradation manner leading to differentiation block in AML. Here, we demonstrate that CDK2-instigated C/EBPα downregulation is actually mediated by SKP2. Mechanistically, we show that CDK2 stabilizes SKP2 by phosphorylating it at Ser64 and thereby potentiates C/EBPα ubiquitination and subsequent degradation in AML cells. Immunoblot experiments showed that CDK2 inhibition downregulated SKP2 levels and concomitantly enhanced C/ EBPα levels in myeloid cells. We further show that while CDK2 promoted C/EBPα ubiquitination and inhibited its protein levels, negatively affected its transactivation potential and DNA binding ability, simultaneous SKP2 depletion abrogated CDK2-promoted ubiquitination and restored C/EBPα expression and function. Taken together, these findings consolidate that CDK2 potentiates SKP2-mediated C/EBPα degradation in AML and targeting CDK2-SKP2 axis can be harnessed for therapeutic benefit in AML.

 Arun Kumar Trivedi [email protected]
1 LSS008 Division of Cancer Biology, CDRI, CSIR-Central Drug Research Institute, Sector-10, Jankipuram Extension, Lucknow, UP 226031, India
2 Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India
3 LSS007 Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow, India

Graphic abstract
Hypothetical model depicts that SKP2-mediated C/EBPα proteasomal degradation is reinforced by CDK2. CDK2 phopsho- rylates SKP2 leading to its enhanced stabilization which in turn exaggerates C/EBPα degradation leading to differentiation arrest in AML.

Keywords C/EBPα · SKP2 · Ubiquitination · Myeloid differentiation · Ubiquitin ligase

Introduction
C/EBPα is founding member of leucine zipper family of transcription factors which regulates myeloid differentia- tion (granulopoiesis in particular) and stem cell homeosta- sis in hematopoietic cells [1–4]. C/EBPα is a tumor sup- pressor in AML for its ability to inhibit growth of Acute Myeloid Leukemia (AML) cells [5]. Because C/EBPα couples myeloid growth arrest with differentiation, loss of C/EBPΑ expression, activity and function is common in AML [6–8]. Besides, C/EBPα mutations are also observed in 8–14% AML patients [9, 10]. Biallelic CEBPα muta- tions are favorable marker of prognostic outcome. Loss of CEBPα expression and function promoting leukemogen- esis have been attributed to multiple mechanisms ranging from CEBPα promoter hypermethylation, regulation at promoter levels, antagonistic protein–protein interactions and more importantly post translational modifications (PTM) [5, 8, 11]. While SUMOylation and phosphoryla- tion of CEBPα are known to inhibit CEBPα function in AML [12], ubiquitination is another PTM that was shown to negatively regulate C/EBPα protein stability. Trib2,
FBW7, E6AP, SKP2 and CDK2 are among some of the proteins that were shown to target C/EBPα for ubiquitin- mediated proteasome degradation [3, 13–18].
Cell cycle exit by myeloid cells is a must for cells to undergo differentiation. While C/EBPα inhibits cell cycle progression by inhibiting E2F [19, 20], Cyclin-depend- ent kinases (CDKs) such as CDK2 regulate normal cell cycle progression. CDK2 in complex with cyclins drives G1 phase progression and entry to S-phase by phospho- rylating Retinoblastoma and contributing to E2F release from Rb-E2F complex [21]. Increased CDK2 expression and activity has previously been indeed demonstrated to promote uncontrolled myeloid cell proliferation and dif- ferentiation arrest [22, 23]. While role of CDK2 in cell cycle progression is widely known, its role in blocking myeloid differentiation in leukemia was recently shown [17, 24]. Ying et al. showed that CDK2 was downregulated through ubiquitin-mediated proteasome in AML cell lines chemically induced to differentiate indicating for its pos- sible role in blocking differentiation [24]. We also recently showed that CDK2 inhibited myeloid differentiation by downregulating C/EBPα through ubiquitin-mediated

proteasome degradation although underlying E3 ligases remained unidentified [17].
Here, we sought to identify underlying E3 ligase through which CDK2 promoted C/EBPα degradation. Although we showed in an independent study that SKP2 inhibited C/ EBPα protein stability in AML, here we show that CDK2- mediated C/EBPα degradation is in fact mediated by SKP2 [18]. Our finding demonstrates that CDK2 stabilizes SKP2 through its phosphorylation at S64 and while CDK2 over- expression inhibits ectopically expressed as well as endog- enous C/EBPα, simultaneous SKP2 depletion/inhibition abrogates CDK2-mediated C/EBPα downregulation.

Materials and methods
Cell culture and transfections

HL-60, THP-1 and U937 cells were cultured in RPMI- 1640 medium supplemented with 10% FBS and 1 × Anti- Anti solution at 37 °C in 5% humidified CO2 incubator. HEK293T was cultured in DMEM high glucose media with 10% FBS and 1 × Anti-Anti and 1 × Sodium pyruvate. Cells were grown at 37 °C in 5% humidified CO2 incubator. Mye- loid cell lines HL-60, THP1, and U937 cells differentiate to granulocyte or monocyte macrophage lineage when induced with suitable differentiating agents. Authentication of cell lines was done through STR profiling on payment basis by DNA forensics laboratory private limited, New Delhi. All transient transfections were performed using Turbofect and lipofectamine2000 transfection reagents according to manu- facturer’s protocol. For knockdown studies, transfection of siSKP2 (pool of 3 oligonucleotides from Santacruz Inc.) or shRNA (combination of 2 shRNA against SKP2 were used in all the knockdown experiments) was achieved using Dher- maFECT transfection reagent as per manufacturer’s proto- col. Transfections in myeloid HL-60 cell line was performed using AMAXA cell Nucleofector kit V (VCA-1003; Lonza) as per manufacturer’s protocol.

Plasmids and antibodies

Expression plasmids for CDK2, Flag-SKP2, pCDNA6- p42C/EBPα-His, HA-Ubiquitin and p(C/EBPα)2TK- luc were previously described [17, 18, 25]. Expres- sion plasmids for pcDNA3-SKP2-HA-S-64,72AA and pcDNA3-SKP2-HA-S-64,72ADD were kind gifts from Dr. Sylvain Meloche [26]. shRNAs (TRCN0000007530, TRCN0000007534 against human SKP2) were purchased from mission shRNA Sigma. Anti-Flag (Clone M2) and HRP-conjugated anti-β-Actin antibodies were purchased from Sigma; anti-HA, anti-C/EBPα from cell signaling
technologies; anti-SKP2 p45 (Clone H-435), and human siSKP2 (sc-36499) were procured from Santa Cruz Biotechnology.

Western blot analysis

Western blotting was performed as previously described [17]. In short, cells were lysed in RIPA lysis buffer [1% (w/w) NP40, 0.5% (w/v) Sodium deoxycholate, 0.1% (w/v) SDS, 0.15 M NaCl, 5 mM EDTA and 1 mM DTT] supple- mented with phosphatase and protease inhibitor cocktails (Sigma). Equal amounts of proteins were resolved on 10% SDS-PAGE, transferred on PVDF membrane and probed with 1:1000 dilution of primary antibody (1:500 for endog- enous proteins) and 1:10,000 for HRP-conjugated secondary antibody. Chemiluminiscence was detected with ECL west- ern blotting detection reagents (Millipore) using LAS 4000 (GE Healthcare). Cells were treated with 10 µM MG132 for 6 h prior to lysate preparation.

Ni–NTA precipitation (His‑purification)

HEK293T cells transiently transfected with C/EBPα and indicated expression plasmids were harvested using lysis buffer (8 M Urea, 100 mM NaH2PO4, 100 mM Tris–Cl; pH 8.0) and incubated with Ni–NTA agarose beads for 4–6 h at RT or O/N at 4 °C. Subsequently beads were washed twice with wash buffer pH 6.0 (8 M Urea, 100 mM NaH2PO4, 100 mM Tris-Cl) and affinity purified proteins were resolved on 10% SDS-PAGE for western blotting.

Electrophoretic mobility shift assay (EMSA)

Gel shift assay was performed with nuclear extracts from HEK293T with indicated transfectants. The oligonucleotides used were derived from the G-CSF receptor promoter [18], the sequences are as follows (C/EBPα binding sites are bold and underlined): Forward strand, 5′-[6 ~ FAM]GATCAG GTGTTGCAATCCCCAG-3′, reverse strand, 5′-[6 ~ FAM] CTGGGGATTGCAACACCTGATC-3′. Annealing of Oli- gonucleotide strands was performed in 1 × Annealing buffer containing 10 mM Tris (pH 7.5), 1 mM EDTA and 50 mM NaCl. Subsequently, EMSA was performed by incubating 20 µg of nuclear extract with 100 nM double-stranded oli- gonucleotides in a 20 µl reaction mixture containing 10 mM Tris (pH 7.5), 50 mM KCl, 2.5 mM MgCl2, 1 mM DTT,
10% Glycerol and 0.5 µg poly(dI-dC) at RT for 30 min. Binding reactions were resolved on a 5% non-denaturing polyacrylamide gel with 0.5 × TBE (89 mM Tris borate and 2 mM EDTA) and electrophoresed at 80 V at 4˚C.

Co‑immunoprecipitation (Co‑IP)
and immunophenotyping analysis through flow cytometer

Co-IP assays were performed as previously described [17, 18, 27]. For immunophenotyping of differentiation surface marker CD11b, myeloid cells U937 and HL60 were either treated with indicated compounds or transiently transfected with equimolar concentrations of two different shRNAs constructs for indicated time points by AMAXA nucleo- fection. Post- transfection, 1 × 106 cells were washed twice with 1 × PBS and incubated with phycoerythrin-conjugated CD11b antibody or phycoerythrin-conjugated IgG isotype control antibody at 37 °C for 30 min in dark. Cells were washed and analyzed by flow cytometry using FACS Calibur flow cytometer (Becton Dickinson, USA).
Luciferase promoter assay

HEK293T cells were seeded one day before transfection at a density of 1 × 105 cells/ml in 24 well plates as previously described [17]. Cells were transfected with pTK(C/EBPα)2- luc promoter (a luciferase expression plasmid containing two copies of a consensus C/EBPα binding site) and expres- sion plasmids for C/EBPα-His, SKP2, CDK2 and shRNAs as indicated. 30 h post transfection, cells were lysed and assayed for luciferase activity using luciferase assay reagent (Promega, Madison, WI). Data presented are means of trip- licate values obtained from representative experiments.
Statistical analysis

Results of representative of three independent experiments. The two-tailed, student’s t-test was used to compare between two groups. Significance was demonstrated by p-value (*).
*p < 0.05, **p < 0.001 and ***p < 0.0001. Densitometric val- ues calculated using MYImage analysis software (Ther- mofisher) below immunoblots indicate expression levels of test proteins normalized with loading control.

Results
CDK2 inhibition downregulates SKP2 and stabilizes C/EBPα

In two independent studies published earlier, we showed that CDK2 and SKP2 (S-phase kinase associated protein 2; an F-box family E3 ubiquitin ligases) targeted C/EBPα for ubiquitin-mediated degradation causing differentiation arrest in acute myeloid cells [17, 18]. While underlying E3 ubiquitin ligase in CDK2-mediated C/EBPα degradation remained elusive, involvement of kinase if any, in E3 ligase
SKP2-mediated ubiquitin–proteasome degradation of C/ EBPα was not explored. Notably, SKP2 in majority of cases targets its substrate for degradation in a phosphorylation- dependent manner [28, 29]. We therefore, used different kinase inhibitors (kinases either known to phosphorylate or interact with C/EBPα) to examine their impact on SKP2- mediated C/EBPα inhibition. Strikingly, CDK2 inhibition maximally mitigated SKP2-mediated C/EBPα downregu- lation indicating for a role of CDK2 in SKP2-mediated C/EBPα protein regulation (Fig. 1a). Interestingly, in our previous study we showed that although CDK2 interacted with C/EBPα, CDK2 interaction with C/EBPα did not seem to have any role in C/EBPα downregulation because a C/ EBPα deletion mutant C/EBPα_Δ137-235 (lacked CDK2 interaction region) was still polyubiquitinated and degraded when coexpressed with CDK2 indicating that CDK2 direct interaction with C/EBPα (nor its phosphorylation by CDK2) was not required for C/EBPα downregulation[17]. Instead, CDK2 seemed to interact with and positively regulate some E3 ubiquitin Ligase that ubiquitinated and degraded C/ EBPα. In order to address if SKP2 could be the underlying E3 ligase in CDK2-mediated C/EBPα downregulation, we ectopically expressed C/EBPα either alone or together with CDK2 and simultaneously depleted SKP2 in HEK293T cells which showed that, as expected, while CDK2 overexpression downregulated C/EBPα, simultaneous SKP2 depletion in CDK2 overexpressed condition mitigated C/EBPα down- regulation (Fig. 1b) confirming that CDK2 downregulated C/EBPα through SKP2. We next examined impact of CDK2 inhibition on levels of SKP2 and C/EBPα in myeloid cells by inhibiting CDK2 through Roscovitine which showed that CDK2 inhibition led to decrease in SKP2 and increase in C/ EBPα protein levels (Fig. 1c) which suggested that CDK2 positively regulated SKP2 to downregulate C/EBPα.
CDK2 phosphorylates and stabilizes SKP2
to potentiate C/EBPα downregulation by SKP2

As we observed CDK2 inhibition by Roscovitine led to decrease in SKP2 levels, we further used CDK2 specific inhibitor NU6140 and yet again observed drastic decrease in SKP2 levels which confirmed that CDK2 indeed posi- tively regulated SKP2 levels (Fig. 2a). Our data are in line with earlier report that showed CDK2 phosphorylated and stabilized SKP2 [26]; we therefore next examined if CDK2 indeed phosphorylated and stabilized SKP2 which in turn could have downregulated C/EBPα. As shown in Fig. 2b, CDK2 overexpression indeed phosphorylated & stabi- lized SKP2 leading to C/EBPα downregulation. In order to consolidate further, we examined C/EBPα levels upon overexpression of phospho-deficient (SKP2-S64,72AA) and constitutively active SKP2 (SKP2-S64,72DD) which showed that constitutively active SKP2 (SKP2-S64,72DD)

Fig. 1 CDK2 inhibition downregulates SKP2 and stabilizes C/ EBPα a Immunoblot shows C/EBPα levels upon ectopic expres- sion of C/EBPα either alone or together with SKP2 treated with different kinase inhibitors (30 µM Roscovitine, 7 µM PD98059 and 10 µM SB216763) as indicated. b Western blot analysis of WCEs of HEK293T cells transfected with HA-C/EBPα alone or together with CDK2 and 25 nM siSkp2. Cells were treated with 10 µM MG132 for 6 h before harvesting. c Immunoblot showing expression of SKP2 and C/EBPα in WCEs of U937 and THP-1 treated with 30 µM Rosco- vitine. β-Actin probed as loading control

indeed exaggerated C/EBPα downregulation (Fig. 2c right panel) confirming that phosphorylation stabilizes SKP2 leading to its enhanced E3 ligase activity.
CDK2, SKP2 and C/EBPα are present in a common immune complex

Having established that CDK2 regulated C/EBPα protein levels through SKP2, we next examined if these three pro- teins existed in same immunocomplex. In order to address this, we ectopically expressed His-C/EBPα either alone or together with Flag-SKP2 and treated the cells with MG132 and Roscovitine as indicated. Whole cell extracts were then used for affinity purification using Ni–NTA columns, affinity precipitated proteins were then resolved on 10% SDS-PAGE and probed with indicated antibodies which showed these proteins (C/EBPα, SKP2 and CDK2) are present in same interactome (Fig. 3a). Expectedly, we observed intensified C/EBPα levels in both MG132 and Roscovitine treated con- ditions. We also confirmed them to be interacting in vivo in myeloid cells through co-immunoprecipitation (Fig. 3b). These data thus demonstrate that SKP2, CDK2 and C/EBPα are indeed part of a common interactome.
SKP2 depletion inhibits CDK2‑instigated C/EBPα ubiquitination

In line with our finding that SKP2 underlies in CDK2-insti- gated C/EBPα downregulation, we next performed in-cell ubiquitination to confirm that SKP2 depletion mitigated CDK2 instigated polyubiquitination of C/EBPα. As shown in Fig. 4, while CDK2 overexpression promoted C/EBPα polyubiquitination leading to its downregulation, simulta- neous depletion of SKP2 abrogated C/EBPα ubiquitina- tion and restored its expression. This data further confirms that CDK2 promoted C/EBPα downregulation through SKP2-mediated ubiquitination and subsequent proteasomal degradation.
SKP2 inhibition abrogates CDK2‑mediated inhibition of C/EBPα transactivation, DNA binding ability and promotes myeloid differentiation

As our data showed that CDK2 inhibited C/EBPα pro- tein levels by potentiating its ubiquitination and proteas- ome-dependent degradation through E3 ligase SKP2. We next examined impact of C/EBPα functions upon SKP2 depletion/inhibition in CDK2 overexpressed conditions. To address this, we examined C/EBPα transactivation potential using C/EBPα-dependent promoter-luciferase reporter assay containing a minimal reporter and two cop- ies of C/EBP binding sites [pTK(C/EBPα)2] which showed while both SKP2 and CDK2 independently inhibited C/ EBPα transactivation function, simultaneous depletion of SKP2 in CDK2 overexpressed condition abrogated CDK2 inhibitory effect on C/EBPα transactivation func- tion (Fig. 5a). Subsequently, Gel-shift assay performed

Fig. 2 CDK2 phosphorylates and stabilizes SKP2 to potenti- ate C/EBPα downregulation by SKP2: a Immunoblot shows levels of SKP2 in WCEs of THP-1 and U937 cells treated with 10 µM NU6140. b WCEs of HEK293T transfected with C/EBPα alone or together with CDK2 were resolved on 10% SDS-PAGE and probed with indicated antibodies. c Immu- noblot shows expression of C/ EBPα upon transfection of Wild type and SKP2 point mutants in WCEs of HEK293T cells

with FAM-labeled oligonucleotides derived from the G-CSF receptor promoter (a C/EBPα target gene) and nuclear extracts of HEK293T cells transiently transfected with C/EBPα alone or together with CDK2 and simultane- ously depleted SKP2 showed that while CDK2 copiously inhibited C/EBPα DNA binding ability, simultaneous SKP2 depletion abrogated inhibitory effect of CDK2 on C/EBPα DNA binding ability (Fig. 5b). Immunoblot in Fig. 5c depicts knock down ability of two different SKP2 shRNAs when transfected either alone or both together
in equal amounts. Equal amounts of both shRNA were used to achieve maximum knockdown efficacy through- out the manuscript. Consistent with abrogation of CDK2 inhibitory effect on C/EBPα transactivation potential and DNA binding ability upon simultaneous SKP2 depletion, we also observed that while pharmacological inhibition of either CDK2 or SKP2 individually promoted U937 differ- entiation, inhibition of the both SKP2 and CDK2 together caused profound differentiation in these U937 cells (Fig. 5d). Furthermore, we also observed that while CDK2

Fig. 3 SKP2 physically associates with C/EBPα and targets it for ubiquitin-mediated proteasome degradation: a Immunoblotting for C/ EBPα, SKP2 and CDK2 following His-purification of C/EBPα with Ni–NTA beads from HEK293T cell lysates transfected with His-C/ EBPα either alone or together with SKP2. b Endogenous C/EBPα

Fig. 4 SKP2 depletion inhibits CDK2 instigated C/EBPα ubiquitina- tion: HEK293T cells were co-transfected with expression plasmids for His-tagged C/EBPα, HA-ubiquitin CDK2 and shSKP2 as indi- cated. C/EBPα was immunoprecipitated using His-tag antibody and immunoblotted with K48-linkage specific ubiquitin, C/EBPα and SKP2 antibodies

and SKP2 RNAi enhanced ATRA-induced differentiation of HL60 cells (differentiates to granulocytes when induced with ATRA), CDK2 overexpression potently inhibited and this CDK2 inhibitory effect abolished when SKP2 was simultaneously depleted (Fig. 5e). Taken together, these data indicate that CDK2 inhibited C/EBPα expression and function by instigating its ubiquitin-mediated proteasome degradation by stabilizing E3 ubiquitin ligase SKP2.
was immunoprecipitated from lysates of U937 cells using a mouse antibody recognizing total C/EBPα protein, followed by immunoblot- ting against C/EBPα (rabbit), SKP2 and CDK2 antibodies. Normal mouse IgG antibody was used as a control for IP

Discussion
In the current study, we focused to identify underlying E3 ubiquitin ligase involved in C/EBPα ubiquitination and deg- radation instigated by CDK2. While in our previous inde- pendent studies, we showed that CDK2 and SKP2 destabi- lized C/EBPα by promoting its ubiquitination, here we show that CDK2 actually stabilizes SKP2 by phosphorylating it at Serin-64 and thus potentiates rapid degradation of C/EBPα by SKP2.
We and others recently emphasized on role of CDK2 in AML pathogenesis where hyperactivation and overexpres- sion of CDK2 in Acute myeloid leukemia has been shown to promote proliferation, survival and differentiation arrest. While we showed that CDK2 destabilized C/EBPα by pro- moting its proteasome-mediated degradation leading to differentiation arrest, CDK2 itself was shown to be down- regulated through ubiquitination in myeloid cells undergoing chemical-induced differentiation as a pre-requisite for dif- ferentiation. Although we showed that CDK2 destabilized C/ EBPα, the underlying E3 ubiquitin ligase remained elusive. In addition, in an independent study we also identified SKP2 as an E3 ligase for C/EBPα, however, no correlation between CDK2 and SKP2 was explored.
SKP2 in majority of cases targets its substrates for ubiquitination that are prior phosphorylated by serine- threonine kinases [28, 29]. For examples, p27 ubiquitin- mediated degradation by SKP2 is promoted upon p27 phosphorylation at Thr187 by Cyclin E-CDK2 complex that drives progression of cells into mitosis [28]. In an effort to identify kinase, if any, involved in SKP2-medi- ated degradation of C/EBPα we stumbled upon CDK2 as CDK2 inhibitor Roscovitine maximally mitigated SKP2- promoted C/EBPα degradation. It was quite intriguing to

Fig. 5 SKP2 inhibition abrogates CDK2-mediated inhibition of C/ EBPα transactivation, DNA binding ability and promotes myeloid differentiation: a Luciferase activity in HEK293T cells post 30 h of transient transfection with pTK(C/EBPα)2-luc reporter construct and indicated amounts of expression plasmids for C/EBPα either alone or together with Flag-SKP2, CDK2 or shSKP2. Data are representative of two independent experiments. Results are given as standard error of mean (+ S.E.M.); *p < 0.05, **p < 0.001, ***p < 0.0001 b Gel-shift assay performed using double-stranded C/EBPα binding site oli- gonucleotides. Equal amounts of nuclear extracts from HEK293T cells transfected with C/EBPα and CDK2 with/without shSKP2 and
control were used. Lane 1 contains free probe only and cold probe
indicates unlabeled oligonucleotides added in excess. c Immunoblot shows knockdown efficiency of two different shSKP2 shRNAs indi- vidually and in combination in equal amounts in HEK293T cells. d U937 cells treated either with NU6140 (10 µM) or SKPin C-1 (10 µM) were stained with PE-conjugated CD11b after 48 h and analyzed under Flow cytometer. e Differentiation marker CD11b expression measured by flow cytometry in HL-60 cells as indicated in conditions with treatment of 1 µM ATRA for 48 h. Numbers in the graphs represent the percentage of CD11b positive cells compared with cells alone. Data are representative of three independent experi- ments

us because, in our previous study where showed CDK2 destabilized C/EBPα protein levels, we observed that CDK2 also downregulated a C/EBPα deletion mutant (C/ EBPα_Δ137-235) that lacked well documented CDK2 interaction region presumably indicated that CDK2 nei- ther directly interacted nor phosphorylated C/EBPα to destabilize it. Instead, CDK2 appeared to regulate some E3 ubiquitin ligase to potentiate C/EBPα degradation.
Consistent with this, we indeed confirmed that CDK2 stabilized SKP2 by phosphorylating it at Ser-64 which in turn profoundly downregulated C/EBPα. In contrary, CDK2 inhibition led to drastic decrease in SKP2 and con- comitant increase in C/EBPα protein levels. Furthermore, simultaneous depletion of SKP2 in CDK2 overexpressed conditions restored C/EBPα levels confirming yet again that SKP2 could be the underlying ubiquitin ligase in CDK2-mediated C/EBPα downregulation. In addition, while CDK2 inhibited C/EBPα transactivation potential, DNA binding ability and differentiation promoting func- tions, simultaneous SKP2 depletion in CDK2 expressed conditions abrogated these inhibitory effects of CDK2 on C/EBPα functions further confirming that CDK2 insti- gated/potentiated C/EBPα ubiquitin-mediated proteasome degradation through SKP2. Because CDK2 and SKP2 are known to drive cell cycle progression while C/EBPα antagonizes and acts as a strong tumor suppressor in AML, C/EBPα profound and rapid downregulation by CDK2 and SKP2 could be required to impart proliferation and sur- vival advantage and block in differentiation in acute mye- loid leukemia. Interestingly, our finding also provides pos- sible mechanism underlying some of the recent findings that showed CDK2 inhibition synergized with ATRA and promoted myeloid differentiation in AML cell lines [30, 31]. Taken together, these findings confirm that CDK2- SKP2 axis targets C/EBPα in AML and targeting this axis could be harnessed for therapeutic benefit in AML.
Acknowledgements Authors also acknowledge technical support pro- vided by Dr. A. L. Vishwakarma (FACS Unit) of Sophisticated and Analytical Instrument Facility of CSIR-CDRI. CDRI communication number for this article is 10247.
Author contributions Study design: AKT; Study conduct: GT, MM, AS, AKS; Data analysis: AKT, GT, AS; Data interpretation: AKT, GT, MM, SS; Drafting manuscript: GT, AKT. Approving final version of manuscript: AKT, GT.
Funding Grant-in-aid (GAP0239) from Lady Tata Memorial Trust (LTMT), Mumbai to Arun Kumar Trivedi and fellowship to Gatha Thacker from Indian Council of Medical Research (ICMR), New Delhi is acknowledged.

Data availability All the materials, data and associated protocols of this study shall be made available to bona fide researcher or reader requests without undue delay or qualifications.
Declarations
Conflict of interest The authors declare that they have no conflict of interest.

References
⦁ Keeshan K, Santilli G, Corradini F, Perrotti D, Calabretta B. Tran- scription activation function of C/EBPalpha is required for induc- tion of granulocytic differentiation. Blood. 2003;102:1267–75.
⦁ Min YM, Zhang H, Giovanni AG, Henry YH, Staber PB, Zhang P, Levantini E, Alberich-Jordà M, Zhang J, Kawasaki A, Tenen DG. C/EBPa controls acquisition and maintenance of adult hemat- opoietic stem cell quiescence. Nat Cell Biol. 2013;15:385–94.
⦁ Pal P, Lochab S, Kanaujiya JK, Kapoor I, Sanyal S, Behre G, Trivedi AK. E6AP, an E3 ubiquitin ligase negatively regulates granulopoiesis by targeting transcription factor C/EBPalpha for ubiquitin-mediated proteasome degradation. Cell Death Dis. 2013;4:e590.
⦁ Avellino R, Delwel R. Expression and regulation of C/EBPalpha in normal myelopoiesis and in malignant transformation. Blood. 2017;129:2083–91.
⦁ Pulikkan JA, Tenen DG, Behre G. C/EBPalpha deregulation as a paradigm for leukemogenesis. Leukemia. 2017;31:2279–85.
⦁ Porse BT, Pedersen TA, Xu X, Lindberg B, Wewer UM, Friis- Hansen L, Nerlov C. E2F repression by C/EBPalpha is required for adipogenesis and granulopoiesis in vivo. Cell. 2001;107:247–58.
⦁ Pabst T, Mueller BU. Transcriptional dysregulation during mye- loid transformation in AML. Oncogene. 2007;26:6829–37.
⦁ Trivedi AK, Pal P, Behre G, Singh SM. Multiple ways of C/ EBPalpha inhibition in myeloid leukaemia. Eur J Cancer. 2008;44:1516–23.
⦁ Pabst T, Mueller BU, Harakawa N, Schoch C, Haferlach T, Behre G, Hiddemann W, Zhang DE, Tenen DG. AML1-ETO downregu- lates the granulocytic differentiation factor C/EBPalpha in t(8;21) myeloid leukemia. Nat Med. 2001;7:444–51.
⦁ Pabst T, Mueller BU, Zhang P, Radomska HS, Narravula S, Schnittger S, Behre G, Hiddemann W, Tenen DG. Dominant- negative mutations of CEBPA, encoding CCAAT/enhancer bind- ing protein-alpha (C/EBPalpha), in acute myeloid leukemia. Nat Genet. 2001;27:263–70.
⦁ Tenen DG. Disruption of differentiation in human cancer: AML shows the way. Nat Rev Cancer. 2003;3:89–101.
⦁ Geletu M, Balkhi MY, Peer Zada AA, Christopeit M, Pulik- kan JA, Trivedi AK, Tenen DG, Behre G. Target proteins of C/EBPalphap30 in AML: C/EBPalphap30 enhances sumoyla- tion of C/EBPalphap42 via up-regulation of Ubc9. Blood. 2007;110:3301–9.
⦁ Bararia D, Kwok HS, Welner RS, Numata A, Sarosi MB, Yang H, Wee S, Tschuri S, Ray D, Weigert O, Levantini E, Ebralidze AK, Gunaratne J, Tenen DG. Acetylation of C/EBPalpha inhibits its granulopoietic function. Nat Commun. 2016;7:10968.
⦁ Bararia D, Trivedi AK, Zada AA, Greif PA, Mulaw MA, Christo- peit M, Hiddemann W, Bohlander SK, Behre G. Proteomic iden- tification of the MYST domain histone acetyltransferase TIP60 (HTATIP) as a co-activator of the myeloid transcription factor C/ EBPalpha. Leukemia. 2008;22:800–7.
⦁ Bengoechea-Alonso MT, Ericsson J. The ubiquitin ligase Fbxw7 controls adipocyte differentiation by targeting C/EBPalpha for degradation. Proc Natl Acad Sci USA. 2010;107:11817–22.
⦁ Keeshan K, He Y, Wouters BJ, Shestova O, Xu L, Sai H, Rod- riguez CG, Maillard I, Tobias JW, Valk P, Carroll M, Aster JC, Delwel R, Pear WS. Tribbles homolog 2 inactivates C/

EBPalpha and causes acute myelogenous leukemia. Cancer Cell. 2006;10:401–11.
⦁ Thacker G, Mishra M, Sharma A, Singh AK, Sanyal S, Trivedi AK. CDK2 destabilizes tumor suppressor C/EBPα expression through ubiquitin-mediated proteasome degradation in acute myeloid leukemia. J Cell Biochem. 2020;121:2839–50.
⦁ Thacker G, Mishra M, Sharma A, Singh AK, Sanyal S, Trivedi AK. E3 ligase SCFSKP2 ubiquitinates and degrades tumor sup- pressor C/EBPα in acute myeloid leukemia. Life Sci. 2020. ⦁ https:// ⦁ doi.⦁ org/10.1016/j.lfs.2020.118041.
⦁ D’Alo F, Johansen LM, Nelson EA, Radomska HS, Evans EK, Zhang P, Nerlov C, Tenen DG. The amino terminal and E2F inter- action domains are critical for C/EBP alpha-mediated induction of granulopoietic development of hematopoietic cells. Blood. 2003;102:3163–71.
⦁ Porse BT, Bryder D, Theilgaard-Monch K, Hasemann MS, Ander- son K, Damgaard I, Jacobsen SE, Nerlov C. Loss of C/EBP alpha cell cycle control increases myeloid progenitor proliferation and transforms the neutrophil granulocyte lineage. J Exp Med. 2005;202:85–96.
⦁ Rubin SM. Deciphering the retinoblastoma protein phosphoryla- tion code. Trends Biochem Sci. 2013;38:12–9.
⦁ Lin Y, Li D, Liang Q, Liu S, Zuo X, Li L, Sun X, Li W, Guo M, Huang Z. miR-638 regulates differentiation and proliferation in leukemic cells by targeting cyclin-dependent kinase 2. J Biol Chem. 2015;290:1818–28.
⦁ Radosevic N, Delmer A, Tang R, Marie JP, Ajchenbaum-Cym- balista F. Cell cycle regulatory protein expression in fresh acute myeloid leukemia cells and after drug exposure. Leukemia. 2001;15:559–66.
⦁ Ying M, Shao X, Jing H, Liu Y, Qi X, Cao J, Chen Y, Xiang S, Song H, Hu R, Wei G, Yang B, He Q. Ubiquitin-dependent deg- radation of CDK2 drives the therapeutic differentiation of AML by targeting PRDX2. Blood. 2018;131:2698–711.
⦁ Trivedi AK, Bararia D, Christopeit M, Peerzada AA, Singh SM, Kieser A, Hiddemann W, Behre HM, Behre G. Proteomic iden- tification of C/EBP-DBD multiprotein complex: JNK1 activates stem cell regulator C/EBPalpha by inhibiting its ubiquitination. Oncogene. 2007;26:1789–801.
⦁ Rodier G, Coulombe P, Tanguay PL, Boutonnet C, Meloche S. Phosphorylation of Skp2 regulated by CDK2 and Cdc14B pro- tects it from degradation by APC(Cdh1) in G1 phase. EMBO J. 2008;27:679–91.
⦁ Lochab S, Pal P, Kanaujiya JK, Tripathi SB, Kapoor I, Bhatt ML, Sanyal S, Behre G, Trivedi AK. Proteomic identification of E6AP as a molecular target of tamoxifen in MCF7 cells. Proteomics. 2012;12:1363–77.
⦁ Carrano AC, Eytan E, Hershko A, Pagano M. SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27. Nat Cell Biol. 1999;1:193–9.
⦁ Kim SY, Herbst A, Tworkowski KA, Salghetti SE, Tansey WP. Skp2 regulates Myc protein stability and activity. Mol Cell. 2003;11:1177–88.
⦁ Shao X, Xiang S, Fu H, Chen Y, Xu A, Liu Y, Qi X, Cao J, Zhu H, Yang B, He Q, Ying M. CDK2 suppression synergizes with all-trans-retinoic acid to overcome the myeloid differentiation blockade of AML cells. Pharmacol Res. 2020;151:104545.
⦁ Rashid A, Duan X, Gao F, Yang M, Yen A. Roscovitine enhances All-trans retinoic acid (ATRA)-induced leukemia cell differen- tiation: novel effects on signaling molecules for a putative Cdk2 inhibitor. Cell Signal. 2020;71:109555.
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