The BTB/POZ transcriptional repressor and candidate oncogene BCL6 is frequently misregulated in B-cell lymphomas. The interface through which the BCL6 BTB domain mediates recruitment of the SMRT, NCoR and BCoR corepressors was recently identified. To determine the contribution of this interface to BCL6 transcriptional and biological properties, we generated a peptide that specifically binds BCL6 and blocks corepressor recruitment in vivo. This inhibitor disrupts BCL6-mediated repression and establishment of silenced chromatin, reactivates natural BCL6 target genes, and abrogates BCL6 biological function in B cells. In BCL6-positive lymphoma cells, peptide blockade caused apoptosis and cell cycle arrest. BTB domain peptide inhibitors may constitute a novel therapeutic agent for B-cell lymphomas.

Summary The BCL6 transcriptional repressor is the most frequently involved oncogene in diffuse large B cell lymphoma (DLBCL). We combined computer-aided drug design with functional assays to identify low molecular weight compounds that bind to the corepressor binding groove of the BCL6 BTB domain. One such compound disrupted BCL6/corepressor complexes in vitro and in vivo, and was observed by X-ray crystallography and NMR to bind the critical site within the BTB groove. This compound could induce expression of BCL6 target genes and kill BCL6-positive DLBCL cell lines. In xenotransplantation experiments, the compound was non-toxic and potently suppressed DLBCL tumors in vivo. The compound also killed primary DLBCLs from human patients.

Transcription factors play a central role in malignant transformation by activating or repressing waves of downstream target genes. Therapeutic targeting of transcription factors can reprogram cancer cells to lose their advantages in growth and survival. The BCL6 transcriptional repressor plays a central role in the pathogenesis of diffuse large B-cell lymphomas (DLBCL) and controls downstream checkpoints, including the p53 tumor suppressor gene. We report that a specific inhibitor of BCL6 called BPI can trigger a p53 response in DLBCL cells. This was partially due to induction of p53 activity and partially due to relief of direct repression by BCL6 of p53 target genes. BPI could thus induce a p53-like response even in the presence of mutant p53. Moreover, sequential BCL6 peptide inhibitors followed by p53 peptide or small-molecule activators provided a more powerful antilymphoma effect than either treatment alone by maximally restoring p53 target gene expression. Therefore, tandem targeting of the overlapping BCL6 and p53 transcriptional programs can correct aberrant survival pathways in DLBCL and might provide an effective therapeutic approach to lymphoma therapy. [Cancer Res 2008;68(9):3361-9]

Detailed analysis of factors governing high affinity antibody-antigen interactions yields important insight into molecular recognition and facilitates the design of functional antibody libraries. Here we describe comprehensive mutagenesis of the light chain complementarity determining regions (CDRs) of HIV-1 antibody D5 (which binds its target, '5-Helix', with a reported K D of 50 pM). Combinatorial scanning mutagenesis libraries were prepared in which contact residues on the D5 light chain were varied among WT side chain identity or alanine. Selection of these libraries against 5-Helix, and then sequence analysis of the resulting population were used to quantify energetic consequences of mutation from wild-type to alanine (ΔΔG Ala-WT ) at each contact position. This analysis revealed several hotspot residues (ΔΔG Ala-WT ≥ 1 kcal/mol) that formed combining site features critical to the affinity of the interaction. Tolerance of D5 light chain residues to alternative mutations was explored with a second library. We found that light chain residues located at the center and at the periphery of the D5 combining site contribute to shape complementarity and electrostatic characteristics. Thus, the affinity of D5 for 5-Helix arises from extended interactions involving both the heavy and light chains of D5. These results provide significant insight for future antibody engineering efforts.The specific recognition of an antigen by an antibody is the central feature of humoral immunity, and serves as the basis for countless therapeutic, research, and diagnostic reagents (1,2). A clear delineation of factors governing high affinity antibody-antigen interactions enhances our understanding of how natural antibodies evolve, and provides information for designing antibody libraries from scratch (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Biochemical and biophysical data on protein-targeting antibodies have shown that, similar to other protein-protein interactions, the energetics of intermolecular interfaces between antibodies and their protein antigens are dominated by a small number of residues on either partner ('hotspots' for binding) (13)(14)(15)(16)(17)(18)(19)(20). Residues in the heavy chain CDRs (HCDRs) constitute the majority of the contact surface (the 'structural paratope') in many protein-antibody interactions, and most antibody engineering efforts have focused on these regions (3,(10)(11)(12). However, in some cases both heavy and light chains form extended contacts with the antigen (13)(14)(15)(16). Mutational analysis on the antibody D1.3, which binds hen egg-white lysozyme (HEL) but can also bind other † This work was funded by Einstein start-up funds, a pilot project grant from the Einstein-Montefiore Center for AIDS Research, and the Arnold and Mabel Beckman Foundation Young Investigators Program. J. S. H. is supported by a National Institutes of Health Biophysics Training Grant (T32-GM008572). * To whom correspondence should be addressed: jon.lai@einstein.yu.edu. . . SUPPORTING INFORMATION AVAILABLEExpression, ...

Optimization of cellulose enzymatic hydrolysis is crucial for cost effective bioethanol production from lignocellulosic biomass. Enzymes involved in cellulose hydrolysis are often inhibited by their end-products, cellobiose and glucose. Efforts have been made to produce more efficient enzyme variants that are highly tolerant to product accumulation; however, further improvements are still necessary. Based on an alternative approach we initially investigated whether recently formed glucose could be phosphorylated into glucose-6-phosphate to circumvent glucose accumulation and avoid inhibition of beta-glucosidase from Bacillus polymyxa (BGLA). The kinetic properties and structural analysis of BGLA in the presence of glucose-6-phosphate (G6P) were investigated. Kinetic studies demonstrated that enzyme was not inhibited by G6P. In contrast, the presence of G6P activated the enzyme, prevented beta glucosidase feedback inhibition by glucose accumulation and improved protein stability. G6P binding was investigated by fluorescence quenching experiments and the respective association constant indicated high affinity binding of G6P to BGLA. Data reported here are of great impact for future design strategies for second-generation bioethanol production.