Therapeutic targeting of the BCL6 oncogene for diffuse large B-cell lymphomas

Abstract

BCL6 is a transcriptional repressor often expressed constitutively in diffuse large B-cell lymphomas (DLBCL) due to mutations of its genomic locus. BCL6 mediates aberrant survival, proliferation, genomic instability and differentiation blockade in DLBCL cells. The biochemical study of BCL6 mediated gene repression has provided the basis for design of agents that inhibit BCL6 and kill lymphoma cells. The repressor activity of the BCL6 BTB domain is particularly well defined from the structural standpoint. Design of inhibitors targeting BCL6 BTB domain protein interaction surfaces appears to be an effective approach, which reactivates important BCL6 target genes and readily kills DLBCL cells. Targeting other domains of BCL6 or using histone deacetylase inhibitors to overcome BCL6 mediated repression may also be useful. Recent studies in DLBCL transcriptional signatures have revealed a subset of DLBCLs that are particularly dependent on BCL6 to maintain their survival and these patients could be candidates for clinical trials of BCL6 inhibitors.

Figure 1. The role of BCL6 in normal and malignant B-cells

Panel A: During normal B-cell maturation, activated B-cells form germinal centers (GC) (shaded circle) in order to form high affinity antibodies. Mature B-cells first become centroblasts, highly proliferating cells in which BCL6 expression is induced. These cells form the dark zone of the GC. Pictured below the centroblast cell is a representation of BCL6 and its three domains, the BTB, RD2 and Zinc fingers. BCL6 contributes to the centroblast phenotype by directly repressing the ATR, CHEK1, TP53 and CDKN1A genes through its BTB domain in order to facilitate proliferation and survival during class switch recombination and somatic hypermutation. BCL6 also represses the PRDM1 gene mostly through its second repression domain in order to block further differentiation. Centroblasts eventually migrate to a more heterogeneous area of the GC called the light zone where they encounter T-cells and follicular dendritic cells (FDC). CD40 signaling by T-cells can block the function of the BCL6 BTB domain by blocking its association with N-CoR and thus de-repress checkpoint genes. This presumably allows B cells damaged during affinity maturation to be weeded out (an apoptotic B-cell is pictured attached to a T-cells). Repression of PRDM1 is independent of the BTB domain, which allows B-cells to sustained blockade of PRDM1 and thus prevents premature differentiation. When CD40 signaling is transient these effects are reversible so that B-cells could maintain the centroblast phenotype and undergo further rounds of affinity maturation. More sustained CD40 signaling can induce IRF4 mediated repression of BCL6 and facilitate terminal differentiation of GC B cells selected by the FDCs into plasma cells or memory cells. GC B cells that have reached this stage in the light zone are called centrocytes. BCL6 can also be downregulated through the ATM pathway via proteolytic degradation when genomic damage reaches a critical level in B-cells. Panel B: Translocations or point mutations of the BCL6 locus can cause it to be expression constitutively and contribute to malignant transformation. Exposure of DLBCL cells to BPI can block the repressor effect of the BCL6 BTB domain and induce expression of ATR, CHEK1, TP53 and CDKN1A resulting in cell death. Downregulation of MTA3 can block the repressor effect of the RD2 and induce PRDM1 resulting in differentiation.

2. Domain structure and partner proteins of BCL6

Panel A: Cartoon representation of BCL6. BCL6 forms a homodimer through the N-terminal BTB domain. The middle region of BCL6 acts as an unstructured linker to its C-terminal zinc fingers, which bind to DNA and other proteins. Panel B: The BTB domain of BCL6 can directly bind with the SMRT, N-CoR and BCoR corepressors. the second repression domain can directly interact with the MTA3 corepressor and also contains motifs through which BCL6 function and stability are regulated by acetylation and phosphorylation. The zinc fingers of BCL6 can interact with the ETO corepressor.

Figure 3. Structural features of the BCL6 BTB domain

Panel A: Ribbon representation of the BCL6 BTB dimer, with one chain in red and one in blue. The C and N termini of each chain are indicated. The linker region in full-length BCL6 continues from the C terminus of each BTB chain. Panel B: The buried interface surface in the BCL6 BTB dimer. The red chain in (A) is shown in surface representation, and the monomer surface that is buried upon dimer formation is colored dark red. An equivalent surface is buried in the blue chain. Panel C: Both BCL6 BTB chains are shown in surface representation, and the SMRT BBD peptide is shown in stick form. The lateral groove surface that contacts the BBD is colored in dark blue and dark red according to the contributing chain. Panel D: Top view of the BCL6 BTB dimer, indicating the charged pocket at the dimer interface. The portions of the charged pocket contributed by each monomer are colored in dark blue and dark red according to the contributing chain.

Figure 4. Potential strategies for therapeutic targeting of BCL6

Panel A: BCL6 expression can be downregulated by siRNA or antisense oligonucleotides. Panel B: Some of the potential ways to inhibit BCL6 functions include 1) blockade of the BCL6 lateral groove by BPI; 2) blockade of the charged pocket, as seems to be the case with Apt48; 3) blocking the interaction of BCL6 with MTA3; 4) inhibiting HDACs so that BCL6 becomes constitutively acetylated, which could reduce its association with MTA3. Since HDACs are also associated with the NuRD and SMRT/N-CoR repression complexes this could further impair BCL6 repressor activity; 5) blockade of the ETO-BCL6 interaction; and 6) blockade of the BCL6-DNA interaction. Panel C: Another way to target BCL6 is to induce reactivation or functional activity of BCL6 target genes. For example, p53 activating peptides or small molecules can induce p53 activity and cause cell death in BCL6 positive lymphoma cells, and chemotherapy drugs can kill lymphoma cells through induction of a p53 response. Combination of the approaches shown in A or B with that shown in C might have enhanced anti-tumor effects.