BCL-2 inhibition with ABT-737 prolongs survival in an NRAS/BCL-2 mouse model of AML by targeting primitive LSK and progenitor cells.

Myelodysplastic syndrome (MDS) transforms into an acute myelogenous leukemia (AML) with associated increased bone marrow (BM) blast infiltration. Using a transgenic mouse model, MRP8[NRASD12/hBCL-2], in which the NRAS:BCL-2 complex at the mitochondria induces MDS progressing to AML with dysplastic features, we studied the therapeutic potential of a BCL-2 homology domain 3 mimetic inhibitor, ABT-737. Treatment significantly extended lifespan, increased survival of lethally irradiated secondary recipients transplanted with cells from treated mice compared with cells from untreated mice, with a reduction of BM blasts, Lin-/Sca-1(+)/c-Kit(+), and progenitor populations by increased apoptosis of infiltrating blasts of diseased mice assessed in vivo by technicium-labeled annexin V single photon emission computed tomography and ex vivo by annexin V/7-amino actinomycin D flow cytometry, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, caspase 3 cleavage, and re-localization of the NRAS:BCL-2 complex from mitochondria to plasma membrane. Phosphoprotein analysis showed restoration of wild-type (WT) AKT or protein kinase B, extracellular signal-regulated kinase 1/2 and mitogen-activated protein kinase patterns in spleen cells after treatment, which showed reduced mitochondrial membrane potential. Exon specific gene expression profiling corroborates the reduction of leukemic cells, with an increase in expression of genes coding for stem cell development and maintenance, myeloid differentiation, and apoptosis. Myelodysplastic features persist underscoring targeting of BCL-2-mediated effects on MDS-AML transformation and survival of leukemic cells.

Charge heterogeneity of monoclonal antibodies by multiplexed imaged capillary isoelectric focusing immunoassay with chemiluminescence detection.

Characterization of charge heterogeneity of recombinant monoclonal antibodies (mAbs) requires high throughput analytical methods to support clone selection and formulation screens. We applied the NanoPro technology to rapidly measure relative charge distribution of mAbs in early stage process development. The NanoPro is a multiplexed capillary-based isoelectric immunoassay with whole-column imaging detection. This assay offers specificity, speed and sensitivity advantages over conventional capillary isoelectric focusing (CIEF) platforms. After CIEF, charge variants are photochemically immobilized to the wall of a short coated capillary. Once immobilized, mAbs are probed using a secondary anti-IgG conjugated with horseradish peroxidase. After flushing away excess reagents, secondary antibodies bound to their targets are then detected by chemiluminescence upon incubation with peroxidase reactive substrates. Charge heterogeneity as determined by chemiluminescence was similar to that measured by conventional CIEF technology with absorbance detection for purified mAbs and contaminated mAbs derived directly from host cellular extract. Upon method optimization, the automated CIEF immunoassay was applied to several mAbs of varying isoelectric points, demonstrating the suitability of NanoPro as a rugged high-throughput product characterization tool. Furthermore, qualification of detection sensitivity, precision, and dynamic range are reported with discussion of its advantages as an alternative approach to rapidly characterize charge variants during process development of mAbs.

Acetylation of Smad2 by the co-activator p300 regulates activin and transforming growth factor beta response.

Transforming growth factor beta (TGFbeta) signals primarily through the Smad proteins to regulate cell growth, differentiation, and extracellular matrix production. Post-translational modifications, such as phosphorylation, play an important role in the regulation of the Smad proteins. TGFbeta signaling results in the phosphorylation of Smad2 and Smad3 that then oligomerize with Smad4 and translocate into the nucleus to initiate transcription of TGFbeta target genes. The initiation of transcription is significantly enhanced by the direct interaction of the Smad complex with p300/CBP (CREB-binding protein), a co-activator with intrinsic acetyltransferase activity. However, how p300/CBP enhances transcription through this interaction is not entirely understood. In this report, we show that Smad2, but not the highly homologous Smad3, can be acetylated by p300/CBP in a ligand-dependent manner. At least three lysine residues, Lys(19), Lys(20), and Lys(39), are required for efficient acetylation of Smad2, as mutations altering these lysines abolished Smad2 acetylation in vivo. This acetylation event is required for the ability of Smad2 to mediate activin and TGFbeta signaling. Mutation of the three key lysine residues did not alter the stability of Smad2 or the ability of Smad2 to form a complex with Smad4 on promoter DNA, but it prevented nuclear accumulation of Smad2 and subsequent TGFbeta and activin responses. Thus, our studies reveal a novel mechanism of modulating Smad2 activity and localization through protein acetylation.