ATM Mutant Chronic Lymphocytic Leukaemia Cells are Chemosensitized by Inhibition of DNA-Dependent Protein Kinase

Abstract 433

Defects in ataxia telangiectasia mutated kinase (ATM) confer poor survival in B-cell chronic lymphocytic leukaemia (CLL). ATM gene deletion (11q23) occurs in approximately 20% of cases, and of these, 38% harbor an ATM mutation on the second allele, resulting in complete loss of ATM function. This prevents signaling of DNA double strand break (DSB) damage and compromises homologous recombinational (HR) repair. Importantly, del(11q) cases without corresponding ATM mutation display wild type ATM activity, with an intact DNA damage response and better outcome than ATM mutated cases¹. Thus, in terms of chemotherapeutic resistance, it has been established that not all del(11q) cases should be placed in one group, since those with wild type ATM function have a distinct response. DNA-dependent protein kinase (DNA-PK) is also important for DSB repair and mediates the alternative non homologous end-joining (NHEJ) pathway. We previously showed that increased DNA-PK activity and expression is frequent in poor prognosis CLL and is associated with shorter survival². We hypothesized that targeting the NHEJ pathway (via DNA-PK inhibition), in ATM deficient CLL cells that already have a defective DNA damage response, would completely compromise the ability of these cells to perform DSB repair, resulting in selective killing of ATM mutant CLL cells.

The ATM status of CLL cases was determined using denaturing HPLC followed by confirmatory direct sequencing. Only those cases with pathogenic mutations (e.g. predicted to cause truncations or change in protein structure, or previously reported in A-T patients) were classed as ‘ATM mutant'. The resulting defects in ATM activity in these cases was confirmed by quantifying phosphorylation of downstream ATM targets (e.g autophosphorylation of ATM (ser1981) or phosphorylation of SMC-1) in lysates of ionizing-radiation-treated cells. Ex vivo cytotoxicity using freshly isolated CLL cells was possible in 77 cases of known ATM status. Cells were exposed to increasing concentrations of Fludarabine in combination with the DNA-PK inhibitor, NU7441 (1μ M), for 72 hr, and viability was assessed using an XTT kit. NU7441 potentiated the cytotoxicity of Fludarabine in 31 cases, including 8 cases with a known ATM mutation. As expected, ATM mutant CLL cells were more drug-resistant than those with wild type ATM, but even highly resistant (>20μ M) ATM mutant cases were resensitized up to 20-fold by NU7441. Pilot data using a similar approach showed ATM mutant cases to be 5-fold more resistant to mitoxantrone, but NU7441 potentiated the cytotoxicity of mitoxantrone in ATM mutant cases (n = 3) and ATM wild type cases (n = 5) by up to 6-fold or 3-fold respectively.

Preliminary data (n =18) using an ex vivo assay dot blot assay to measure DNA-PK activity correlated DNA-PK activity to expression of the DNA-PK catalytic subunit as determined by Western blotting. Furthermore, DNA-PK activity was significantly higher in ATM mutant, compared to wild type CLL cells (p = 0.008). Current studies are exploring the formation and longevity of γH2AX foci in ATM mutant CLL cells treated with DNA damaging agents, and the role of DNA-PK in repair of DSB in these cells. Also, the link between DNA-PK activity and ATM mutation will be further examined as it provides a possible mechanism and proof of concept of increased sensitization by DNA-PK inhibitors. These results suggest that DNA-PK inhibition can sensitize poor prognosis, ATM mutant CLL cells to chemotherapeutics. Our data are consistent with the concept of synthetic lethality, where tumor cells harboring a DNA repair defect can be killed by targeting the compensatory DNA repair pathway, and suggest a group of patients that may benefit from this combination.

1 Austen et al., Journal of Clin. Oncol. 2008

2 Willmore et al., Clin. Can. Res. 2008