2.14 The Kinetics of Phosphorylation of the CD5 Regulatory Domain in Chronic Lymphocytic Leukemia B Cells Reflects Ongoing Activation of the B-Cell Receptor

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gene transcription. Similarly, the ataxia telangiectasia mutated kinase (ATM), which senses double strand breaks, is required for DNA damage–induced activation of NFB in breast cancer cells. Furthermore, other reports showed that the DNA damage–signalling enzyme, DNA-dependent protein kinase (DNA-PK) activates NFB by regulating MEK/ERK signalling, the IKK complex, and the p50 subunit. We previously demonstrated that inhibition of DNA-PK chemosensitises CLL cells and that DNA-PK is highly expressed in CLL cells from patients with a poor prognosis. These data collectively led us to hypothesise that, like ATM, part of the DNA-PK–mediated DNA damage response involves regulation of NFB activity. First, we quantified p50, p65, p52, c-rel, and Rel-B subunits in 68 CLL samples (standardised enzyme-linked immunosorbent assay) to determine their role in disease progression and correlation with prognostic markers and resistance. High levels of p50 and p65 subunits correlated with shorter overall survival (hazard ratio 5.8 and 5.7, respectively) and identified patients harbouring del(17p) or del(11q). High levels of p52 and c-Rel had no significant effect on outcome, but in 17 cases of known ATM mutation status, Rel-B levels were significantly higher (p 0.005) in patients with ATM mutations. Increased NFB activity correlated with ex vivo chemoresistance (XTT assay) to fludarabine (p 0.001), chlorambucil (p 0.0001), and mitoxantrone (p 0.03). Of note, in 4 cases, drug-induced activity of the p50 and p65 NFB subunits in fresh CLL cells was abrogated by at least 50% by DNA-PK inhibition (using 1 M of NU7441), indicating that catalytic activity of DNAPKcs is required for drug-induced NFB signalling. In support of this concept, human glioblastoma cells deficient in DNA-PKcs (M059J) were 3-fold more sensitive to the NFB antagonist, BAY 11-7082, than the DNA-PKcs–proficient counterpart (M059J Fus1). These data indicate that in addition to its role in double strand break repair, DNA-PKcs can mediate chemosensitisation by decreasing NFB activity. Pilot data using chromatin immunoprecipitation indicate that NFB subunits are capable of binding to the promoter region of DNA-PKcs, and future studies using knockdown of DNA-PKcs will elucidate the role of DNA-PK on NFB– dependent gene transcription. Collectively, these data suggest that targeting DNA damage activated NFB activity represents a novel approach to overcoming chemoresistance in patients with CLL who have increased levels of the p65 and p50 NFB subunits.