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Many tumor cells exhibit a disturbed intracellular redox state resulting in higher levels of reactive oxygen species (ROS). As these contribute to tumor initiation and sustenance, catalytic redox agents combining significant activity with substrate specificity promise high activity and selectivity against oxidatively stressed malignant cells. We describe here the design and synthesis of novel organochalcogen based redox sensor/effector catalysts. Their selective anticancer activity at submicromolar and low micromolar concentrations was established here in a range of tumor entities in various biological systems including cell lines, primary tumor cell cultures, and animal models. In the B-cell derived chronic lymphocytic leukemia (CLL), for instance, such compounds preferentially induce apoptosis in the cancer cells while peripheral blood mononuclear cells (PBMC) from healthy donors and the subset of normal B-cells remain largely unaffected. In support of the concept of sensor/effector based ROS amplification, we are able to demonstrate that underlying this selective activity against CLL cells are pre-existing elevated ROS levels in the leukemic cells compared to their nonmalignant counterparts. Furthermore, the catalysts act in concert with certain chemotherapeutic drugs in several carcinoma cell lines to decrease cell proliferation while showing no such interactions in normal cells. Overall, the high efficacy and selectivity of (redox) catalytic sensor/effector compounds warrant further, extensive testing toward transfer into the clinical arena.

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...

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Functional inactivation of transcription factors in hematopoietic stem cell development is involved in the pathogenesis of acute myeloid leukemia (AML). Stem cell regulator C/enhancer binding protein (EBP)a is among such transcription factors known to be inactive in AML. This is either due to mutations or inhibition by protein-protein interactions. Here, we applied a mass spectrometry-based proteomic approach to systematically identify putative co-activator proteins interacting with the DNA-binding domain (DBD) of C/EBP transcription factors. In our proteomic screen, we identified c-Jun N-terminal kinase (JNK) 1 among others such as PAK6, MADP-1, calmodulin-like skin proteins and ZNF45 as proteins interacting with DBD of C/EBPs from nuclear extract of myelomonocytic U937 cells. We show that kinase JNK1 physically interacts with DBD of C/EBPa in vitro and in vivo. Furthermore, we show that active JNK1 inhibits ubiquitination of C/EBPa possibly by phosphorylating in its DBD. Consequently, JNK1 prolongs C/EBPa protein half-life leading to its enhanced transactivation and DNA-binding capacity. In certain AML patients, however, the JNK1 mRNA expression and its kinase activity is decreased which suggests a possible reason for C/EBPa inactivation in AML. Thus, we report the first proteomic screen of C/EBP-interacting proteins, which identifies JNK1 as positive regulator of C/EBPa.

Key Points• CD44 expression in CLL is micromilieu instructed and promotes leukemic cell survival, which can be antagonized by CD44 antibodies.• As a surface coreceptor, CD44 supports leukemogenesis by modulating stimuli of MCL1 expression (eg, B-cell receptor signals).The cell-surface glycoprotein CD44 is expressed in chronic lymphocytic leukemia (CLL), but its functional role in this disease is poorly characterized. We therefore investigated the contribution of CD44 to CLL in a murine disease model, the Em-TCL1 transgenic mouse, and in CLL patients. Surface CD44 increased during murine CLL development. CD44 expression in human CLL was induced by stimulation with interleukin 4/soluble CD40 ligand and by stroma cell contact. Engagement of CD44 by its natural ligands, hyaluronic acid or chondroitin sulfate, protected CLL cells from apoptosis, while anti-CD44 small interfering RNAs impaired tumor cell viability. Deletion of CD44 during TCL1-driven murine leukemogenesis reduced the tumor burden in peripheral blood and spleen and led to a prolonged overall survival. The leukemic cells from these CD44 knockout animals revealed lower levels of antiapoptotic MCL1, a higher propensity to apoptosis, and a diminished B-cell receptor kinase response. The inhibitory anti-CD44 antibodies IM7 and A3D8 impaired the viability of CLL cells in suspension cultures, in stroma contact models, and in vivo via MCL1 reduction and by effector caspase activation. Taken together, CD44 expression in CLL is mediated by the tumor microenvironment. As a coreceptor, CD44 promotes leukemogenesis by regulating stimuli of MCL1 expression. Moreover, CD44 can be addressed therapeutically in CLL by specific antibodies. (Blood. 2013;121(20):4126-4136)

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.

IntroductionBalanced translocations that lead to expression of aberrant fusion proteins are a hallmark of acute leukemias. 1 Many of these fusion proteins function as aberrant transcription factors that are an initiating event in leukemogenesis. 2 Some translocations, eg those involving the MLL gene can lead to phenotypically diverse forms of leukemia, whereas the t(15;17) uniformly leads to acute promyelocytic leukemia. The reasons why some translocations are leukemia-type specific, whereas others are not, is unknown.Histone modifications such as methylation of histone H3 at lysine 4 and 9 and acetylation of histone H3 are closely linked to the transcriptional activation status. In t(15;17), the chimeric PML-RAR␣ fusion protein has been shown to recruit corepressors such as DAXX, histone deacetylase (HDAC) activity, DNA methyltransferase activity, and the SUV39H1 histone methyltransferase to RAR␤2, the most extensively studied target gene. [3][4][5][6][7] PML-RAR␣ also interacts with histone deacetylase 1. 8 PML-RAR␣ homodimerization has been shown to relax the relatively stringent RAR␣ DNA binding specificity. 9,10 This gain of function is supposed to lead to many additional genomic binding sites that are not well defined. As a consequence, virtually all direct genomic targets of PML-RAR␣ are currently unknown. In addition, on a global level, the mechanistic alterations occurring at PML-RAR␣ target genes remain to be clarified.On a phenotypic level, the PML-RAR␣ fusion protein blocks differentiation and apoptosis and enhances self-renewal. 11,12 In mouse models, PML-RAR␣ induces a disease similar to acute promyelocytic leukemia (APL). 13,14 Microarray analyses elucidated several leukemogenic mechanisms and pathways. [15][16][17][18][19] For example, PML-RAR␣ induces activation of the Wnt signaling pathway. 17 Also, PML-RAR␣ alters the apoptotic response and expression of differentiation genes. 15 However, these studies do not distinguish between direct and indirect effects on gene expression. This knowledge is crucial to understand the mechanistic implications of PML-RAR␣ and to elucidate the reasons for the unique phenotype that is associated with its activities.The possibilities to understand transcription factor functions has recently been significantly improved by genome-wide approaches that identify target genes in vivo using Chromatin immunoprecipitation (ChIP)-Chip approaches. 20,21 In addition, the ability to use this method to map epigenetic modifications such as histone acetylation at promoters known to bind the transcription factor allows for the identification of the functional consequences of transcription factor binding to its genomic targets. 22,23 Using ChIP-chip analyses, we identified 372 direct PML-RAR␣ genomic targets and show that PML-RAR␣ induces heterochromatin formation on virtually all of its identified target genes. Several of the identified genes are known tumor suppressors and for one of the novel genes (S100P), we show a potential role in the PML-RAR␣-associated block in differenti...