CCAAT/enhancer-binding protein ␣ (C/ EBP␣) is a critical regulator for early myeloid differentiation. Mutations in C/EBP␣ occur in 10% of patients with acute myeloid leukemia (AML), leading to the expression of a 30-kDa dominantnegative isoform (C/EBP␣p30). In the present study, using a global proteomics approach to identify the target proteins of C/EBP␣p30, we show that Ubc9, an E2-conjugating enzyme essential for sumoylation, is increased in its expression when C/EBP␣p30 is induced. We confirmed the increased expression of Ubc9 in patients with AML with C/EBP␣p30 mutations compared with other subtypes. We further confirmed that the increase of Ubc9 expression was mediated through increased transcription. Furthermore, we show that Ubc9-mediated enhanced sumoylation of C/EBP␣p42 decreases the transactivation capacity on a minimal C/EBP␣ promoter. Importantly, overexpression of C/EBP␣p30 in granulocyte colony-stimulating factor (G-CSF)-stimulated human CD34 ؉ cells leads to a differentiation block, which was overcome by the siRNA-mediated silencing of Ubc9. In summary, our data indicate that Ubc9 is an important C/EBP␣p30 target through which C/EBP␣p30 enhances the sumoylation of C/EBP␣p42 to inhibit granulocytic differentiation. IntroductionThe transcription factor CCAAT/enhancer-binding protein ␣ (C/ EBP␣) is crucial for granulocytic differentiation. [1][2][3] Alterations of the function of C/EBP␣ are a common feature of leukemic cells. 4,5 It was discovered in 10% of patients with acute myeloid leukemia (AML) that the CEBPA gene is mutated. 6,7 These mutations are found in AMLs with a myeloblast phenotype (French-AmericanBritish [FAB]-M1 and -M2 subtypes). The mutated gene results in the predominant expression of a 30-kDa protein initiated at an internal AUG start codon. This mutated isoform lacks the Nterminal transactivation domain 1 (TAD1). However, it possesses the intact bZIP protein-protein interaction domain and can interact with activators and repressors that affect its biological roles. The mutated 30-kDa isoform has been shown to act in a dominantnegative manner over the wild-type isoform. 5 The ratio of p30/p42 is critical for a physiologic granulopoiesis. 5,8 In contrast to C/EBP␣p42, C/EBP␣p30 fails to induce myeloid cell differentiation. It inhibits the expression of the endogenous granulocyte colony-stimulating factor (G-CSF) receptor and leads to an enhanced proliferation. 9,10 Recently, it was reported that C/EBP␣p30 directly interacts with the BCL2 promotor to fulfill this role. 11 Relatively little is understood about how C/EBP␣p30 exerts its dominant-negative effect over C/EBP␣p42 and how it inhibits C/EBP␣p42 during normal myeloid lineage development. We applied high-throughput proteomics to identify the target proteins of C/EBP␣p30. In our screen, we identified the ubiquitinconjugating enzyme (Ubc9) as a novel target of C/EBP␣p30.Ubc9 is an essential E2 enzyme required for small ubiquitinrelated modifier (SUMO) conjugation, or sumoylation. 12,13 Ubc9 is known to play a central role in su...

The transcription factor CCAAT enhancer-binding protein a (C/EBPa) has an important role in granulopoiesis. The tumor suppressor function of C/EBPa is shown by the findings that loss of expression or function of C/EBPa in leukemic blasts contributes to a block in myeloid cell differentiation and to leukemia. C/EBPa mutations are found in around 9% of acute myeloid leukemia (AML) patients. The mechanism by which the mutant form of C/EBPa (C/EBPa-p30) exerts a differentiation block is not well understood. By using a proteomic screen, we have recently reported PIN1 as a target of C/EBPa-p30 in AML.In the present study, we show that C/EBPa-p30 induces PIN1 expression. We observed elevated PIN1 expression in leukemic patient samples. Induction of C/EBPa-p30 results in recruitment of E2F1 in the PIN1 promoter. We show that the inhibition of PIN1 leads to myeloid differentiation in primary AML blasts with C/EBPa mutations. Overexpression of PIN1 in myeloid cells leads to block of granulocyte differentiation. We also show that PIN1 increases the stability of the c-Jun protein by inhibiting cJun ubiquitination, and c-Jun blocks granulocyte differentiation mediated by C/EBPa. Our data suggest that the inhibition of PIN1 could be a potential strategy of treating AML patients with C/EBPa mutation.

Acute myeloid leukaemia (AML) is characterized by specific cytogenetic aberrations that are strong determinants of prognostic outcome and therapeutic response. Because the pathological outcome of AML patients with cytogenetic abnormalities differs considerably, we hypothesized that their proteome may also differ specifically in their expression pattern, protein interaction pathways and post-translational modifications (PTM). We performed this study using 42 AML patients diagnosed for various cytogenetic abnormalities based on two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MS) and MSMS tandem MS. We could identify significant differences in the proteome and PTM of peptides, later confirmed by other methods, between cytogenetic groups. The interactome analysis based on computational bioinformatics reveals major regulating networks: MAPK8 and MYC for complex aberrant karyotype, TP53 for t(8;21), TP53-MYC-PRKAC for 11q23 and JUN and MYC for Inv(16). Further, we analysed 42 MS spectra representative of hnRNPH1, calreticulin and hnRNPA2/B1 in a peak explorer, which reveals a cytogenetic-specific PTM of b-O-linked N-acetyl glucosamine (O-GlcNAc) of hnRNPH1 in AML patients with 11q23 translocation, an acetylation of calreticulin in t(8;21) translocation and methylation of hnRNPA2/B1 in patients with translocations of t(8;21) and inv(16). This report may lead to a new thinking about AML pathogenesis, as differences at PTM level could be used to distinguish different subtypes of AML.

The transcription factor CCAAT/enhancer binding protein a (C/EBPa) is important in the regulation of granulopoiesis and is disrupted in human acute myeloid leukemia. In the present study, we sought to identify novel C/EBPa interacting proteins in vivo through immunoprecipitation using mass spectrometrybased proteomic techniques. We identified Max, a heterodimeric partner of Myc, as one of the interacting proteins of C/EBPa in our screen. We confirmed the in vivo interaction of C/EBPa with Max and showed that this interaction involves the basic region of C/EBPa. Endogenous C/EBPa and Max, but not Myc and Max, colocalize in intranuclear structures during granulocytic differentiation of myeloid U937 cells. Max enhanced the transactivation capacity of C/EBPa on a minimal promoter. A chromatin immunoprecipitation assay revealed occupancy of the human C/EBPa promoter in vivo by Max and Myc under cellular settings and by C/EBPa and Max under retinoic acid induced granulocytic differentiation. Interestingly, enforced expression of Max and C/EBPa results in granulocytic differentiation of the human hematopoietic CD34 þ cells, as evidenced by CD11b, CD15 and granulocyte colony-stimulating factor receptor expression. Silencing of Max by short hairpin RNA in CD34 þ and U937 cells strongly reduced the differentiation-inducing potential of C/EBPa, indicating the importance of C/EBPa-Max in myeloid progenitor differentiation. Taken together, our data reveal Max as a novel co-activator of C/EBPa functions, thereby suggesting a possible link between C/EBPa and Myc-Max-Mad network.

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