Abstract
Poor prognosis B-cell chronic lymphocytic leukemia (CLL) is characterised by del(17p), del(11q) and unmutated IgVH genes. Mutational inactivation of p53 and ataxia telangiectasia-mutated kinase (ATM) are more frequent in these patients and confer drug-resistance. Over-expression of DNA-dependent protein kinase (DNA-PK), the enzyme that mediates DNA double strand break (DSB) repair via non homologous end joining (NHEJ), also correlates with chemo-resistance. Thus, alterations in DNA damage signalling pathways are associated with poor risk CLL. We have shown that DNA-PK is a new therapeutic target in CLL1, and are evaluating the efficacy of novel small molecule inhibitors of DNA-PK in ex vivo studies using leukemic lymphocytes from a well-characterised cohort of CLL patients (n=85). We hypothesised that targeting DNA-PK would inhibit NHEJ and thus sensitise CLL cells to drug-induced DNA damage.
NU7441 and KU-0060648 are potent small molecule inhibitors of DNA-PK, developed in collaboration with KuDOS Pharmaceuticals (Cambridge, UK). Lymphocytes were treated with fludarabine, chlorambucil, and Topoisomerase II poisons (mitoxantrone, etoposide, doxorubicin) in the presence or absence of NU7441 (1 μM) or KU-0060648 (0.2 mM). There was a concentration-dependent decrease in viability in response to single agent treatment (XTT/apoptosis assays) that was potentiated in the presence of a DNA-PK inhibitor. For example, 14/18 cases tested with mitoxantrone (currently in clinical trials) were sensitised by NU7441. Measurement of γH2AX foci formation (a surrogate marker for DSB) after Mitoxantrone treatment showed foci formation within 3 hr (n=4), which was maximally potentiated at 24hr following co-incubation with NU7441, implicating DNA-PK as a mediator of DSB repair following drug treatment. Stratification by karyotypic status demonstrated striking results. Although del(17p) cases were more resistant to mitoxantrone (mean LC50 1.2 mM ± 0.2) compared to del(13q) cases (mean LC50 0.4 mM ± 0.03), they had the greatest sensitization (7–13 fold) to Mitoxantrone by NU7441 (p=0.0006), indicating the particular effectiveness of this combination in del(17p) cases. Consistent with this observation, DNA-PK expression (Western blot and activity assays) was highest in del(17p) cases, confirming the utility of this novel drug combination. Whereas Topoisomerase IIα expression was negligible (Western blotting), Topoisomerase IIβ expression varied 3-fold. RT PCR analyses are underway to further study expression of DNA-PK and Topoisomerase II in this cohort. Taken together, these data show that use of a DNA-PK inhibitor increases the therapeutic index of drugs currently used to treat CLL and identify a targeted and novel approach for poor prognosis disease.
DNA-Dependent Protein Kinase Is a Therapeutic Target and an Indicator of Poor Prognosis in B-Cell Chronic Lymphocytic Leukemia