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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.