Abstract
The BCL6 (B-Cell-Lymphoma-6) transcriptional repressor is a critical oncogene in B-cell lymphomas and is required for establishment of germinal centers (GCs) by normal B-cells. However, the mechanisms by which BCL6 licenses GC formation and lymphomagenesis remain unknown. To characterize this mechanism we identified BCL6 target genes through several methods. Most notable among these was ATR, a master DNA damage response mediator. We showed that primary BCL6-expressing GC centroblasts purified from human tonsils do not express ATR, do not activate downstream targets of ATR (Chk1) and exhibit an overall attenuated DNA damage checkpoint response (as shown in COMET assays, H2AX phosphorylation assays, and other methods). ATR expression, the activation of ChK1, and the DNA damage phenotype were fully rescued by BCL6 loss of function (induced by shRNA or by a specific BCL6 inhibitor that we designed). BCL6 expressing DLBCL cell lines exhibited the same BCL6-dependent DNA damage response phenotype. This could be attributed almost entirely to ATR since ectopic expression of ATR could restore DNA damage sensing even in the presence of BCL6. Reciprocally, loss of ATR in BCL6 depleted B-cells could completely prevent them from restoring their damage response pathways. These effects are independent of p53, which in contrast to ATR is expressed in primary centroblasts and in many primary DLBCL cases. Gain of function experiments with BCL6 in isolated human tonsilar naïve B-cells could induce this same damage phenotype in an ATR dependent/p53 independent manner. Most remarkably, we discovered that CD40 signaling, which occurs once centroblasts mature into centrocytes in the GC light zone, rescues ATR from BCL6 mediated repression by signaling to the BCL6 repression complex through NFKB. This causes the SMRT and N-CoR corepressors to be released from BCL6 repressor complexes associated with the ATR promoter, induces ATR expression, its downstream target ChK1 activation and restores DNA damage sensing. As a consequence, B-cells that have undergone extensive damage (as a by-product of antibody affinity maturation and intense proliferation) can now undergo apoptosis, while those with lower level damage undergo cell cycle arrest, DNA repair, and further differentiation. We thus described a molecular feedback loop through which BCL6 mediates GC formation by directly repressing ATR and inducing a DNA damage checkpoint deficient phenotype, and through which subsequent CD40 signaling rescues this phenotype by disrupting the BCL6 repression complex. We showed that sustained BCL6 expression leads to DNA misrepair and genomic instability in primary B cells consistent with its role in lymphomagenesis. Reciprocally, therapeutic targeting of BCL6 synergized with chemotherapy and radiation to kill DLBCL cells (by restoring DNA damage checkpoints) and would thus be expected to improve therapeutic outcomes of DLBCL patients.
Increased Radioresistance and Accelerated B Cell Lymphomas in Mice with Mdmx Mutations that Prevent Modifications by DNA-Damage-Activated Kinases
