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BCR-Abl is a driver oncogene that causes chronic myeloid leukemia and a subset of acute lymphoid leukemias. Although tyrosine kinase inhibitors provide an effective treatment for these diseases, they generally do not kill leukemic stem cells, the cancer-initiating cells that compete with normal hematopoietic stem cells for the bone marrow niche. New strategies to target cancers driven by BCR-Abl are therefore urgently needed. We performed a small molecule screen based on competition between isogenic untransformed cells and BCR-Abl-transformed cells, and identified several compounds that selectively impair the fitness of BCR-Abl-transformed cells. Interestingly, systems-level analysis of one of these novel compounds, DJ34, revealed that it induced depletion of c-Myc and activation of p53. DJ34-mediated c-Myc depletion occurred in a wide range of tumor cell types, including lymphoma, lung, glioblastoma, breast cancer, and several forms of leukemia, with primary leukemic stem cells being particularly sensitive to DJ34. Further analyses revealed that DJ34 interferes with c-Myc synthesis at the level of transcription, and we provide data showing that DJ34 is a DNA intercalator and topoisomerase II inhibitor. Physiologically, DJ34 induced apoptosis, cell cycle arrest and cell differentiation. Taken together, we have identified a novel compound that dually targets c-Myc and p53 in a wide variety of cancers, and with particularly strong activity against leukemic stem cells.Increasing evidence emphasizes the importance of chemokines and chemokine receptors as regulators of bone remodeling. The C-C chemokine receptor 3 (CCR3) is dramatically up-regulated during osteoclastogenesis but the role of CCR3 in osteoclast formation and bone remodeling in adult mice is unknown. Herein, we used bone marrow macrophages (BMM) derived from adult male CCR3-proficient and -deficient mice to study the role of CCR3 in osteoclast formation and activity. CCR3 deficiency was associated with formation of giant hypernucleated osteoclasts, enhanced bone resorption when cultured on bone slices and altered mRNA expression of related chemokine receptors and ligands. Additionally, primary mouse calvarial osteoblasts isolated from CCR3-deficient mice showed increased mRNA expression of the osteoclast activator related gene, receptor activator of nuclear factor kappa-B ligand (Rankl), and osteoblast differentiation associated genes. Micro-computed tomography analyses of femurs from CCR3-deficient mice revealed a bone phenotype that entailed less cortical thickness and volume. Consistent with our in vitro studies, the number of osteoclasts did not differ between the genotypes in vivo Moreover, an increased endo-cortical osteoid mineralization rate and higher trabecular and cortical bone formation rate was displayed in CCR3-deficient mice. Collectively, our data show that CCR3 deficiency influences osteoblast and osteoclast differentiation and that it is associated with thinner cortical bone in adult male mice.Proteins are modulated by a variety of posttranslational modifications including methylation. Despite its importance, the majority of protein methylation modifications discovered by mass spectrometric analyses are functionally uncharacterized, partly due to the difficulty in obtaining reliable methylsite-specific antibodies. To elucidate how functional methylsite-specific antibodies recognize the antigens and lead to development of novel method to create such antibodies, we use an immunized library paired with phage display to create rabbit monoclonal antibodies recognizing trimethylated Lys260 of MAP3K2 as a representative substrate. learn more We isolated several methylsite-specific antibodies which contained unique complementarity determining region sequence. We characterized the mode of antigen recognition by each of these antibodies using structural and biophysical analyses, revealing the molecular details, such as binding affinity toward methylated / unmethylated antigens and s structural motif that is responsible for recognition of methylated lysine residue, by which each antibody recognized the target antigen. In addition, the comparison with the results of western blotting analysis suggests a critical antigen recognition mode to generate cross-reactivity to protein and peptide antigen of the antibodies. Computational simulations effectively recapitulated our biophysical data, capturing the antibodies of differing affinity and specificity. Our exhaustive characterization provides molecular architectures of functional methylsite-specific antibodies and thus should contribute to the development of a general method to generate functional methylsite-specific antibodies by de novo design.Glycoconjugates play a central role in several cellular processes and alteration in their composition is associated with numerous human pathologies. Substrates for cellular glycosylation are synthesized in the hexosamine biosynthetic pathway, which is controlled by the glutaminefructose-6-phosphate amidotransfera-se (GFAT). Human isoform 2 GFAT (hGFAT2) has been implicated in diabetes and cancer; however, there is no information about structural and enzymatic properties of this enzyme. Here, we report a successful expression and purification of a catalytically active recombinant hGFAT2 (rhGFAT2) in E. coli cells fused or not to a HisTag at the C-terminal end. Our enzyme kinetics data suggest that hGFAT2 does not follow the expected ordered bi-bi mechanism, and performs the glucosamine-6-phosphate synthesis much more slowly than previously reported for other GFATs. In addition, hGFAT2 is able to isomerize fructose-6-phosphate into glucose-6-phosphate even in the presence of equimolar amounts of glutamine, which results in unproductive glutamine hydrolysis. Structural analysis of a three-dimensional model of rhGFAT2, corroborated by circular dichroism data, indicated the presence of a partially structured loop in the glutaminase domain, whose sequence is present in eukaryotic enzymes but absent in the E. coli homolog. Molecular dynamics simulations suggest that this loop is the most flexible portion of the protein, and plays a key role on conformational states of hGFAT2. Thus, our study provides the first comprehensive set of data on the structure, kinetics and mechanics of hGFAT2, which will certainly contribute to further studies on the (patho)physiology of hGFAT2.