Targeting DNA-PK and ATM Increases Sensitivity to Chlorambucil and Fludarabine in Chemoresistant and p53-Defective B-CLL Cells.

A subgroup of B-cell chronic lymphocytic leukemia (CLL) patients develop a more aggressive form of the disease characterised by ZAP70 expression and unmutated IgVH genes. Mutational inactivation of p53 and ataxia telangiectasia-mutated kinase (ATM) are frequent in these patients and confer chemo-resistance. Overexpression of DNA-dependent protein kinase (DNA-PK) correlates with clinical resistance to Chlorambucil (CLB). Thus, alterations in DNA damage signaling pathways are associated with drug resistance. We previously demonstrated that inhibition of DNA-PK sensitizes leukemia cell lines to DNA topoisomerase II poisons and ionising radiation (IR) by inhibition of non-homologous end joining of DNA double strand breaks (DSB). Loss of ATM function compromises the complementary DSB repair pathway, homologous recombination. We hypothesised that repair of DNA damage induced by CLB and Fludarabine may be mediated in part via DNA-PK and ATM and that inhibiting these enzymes would sensitize CLL cells to these agents.

NU7441 and KU55933 are potent small molecule inhibitors of DNA-PK and ATM respectively, developed in collaboration with KuDOS Pharmaceuticals (Cambridge, UK). We used these inhibitors in ex-vivo drug combination experiments on CLL lymphocytes derived from a cohort of 60 patients. Lymphocytes were treated with CLB, Fludarabine, Cyclophosphamide, Mitoxantrone or Etoposide in the presence or absence of NU7441 (1μM) or KU55933 (10μM). We observed a concentration-dependent decrease in viability in response to these drugs (XTT assay), and in 70% of cases this decrease was potentiated in the presence of NU7441 (e.g. CLB 2–20 fold potentiation, Fludarabine 2–14 fold). Drug induced caspase 3/7 activity was further increased by NU7441, indicating that DNA-PK inhibition increased cell death via apoptosis. Early data shows that KU55933 also sensitizes tumors to these agents. Increased drug-sensitivity by NU7441 or KU-55933 was independent of disease stage, progression or CD38 status.

We quantified γH2AX foci formation (a surrogate marker for DSB) after drug treatment. Fludarabine induced γH2AX foci formation (7 tumors), and this novel observation suggests that Fludarabine induces DSB in quiescent CLL cells. NU7441 increased the number of fludarabine-induced γH2AX foci, implicating DNA-PK as a mediator of DSB repair following Fludarabine treatment. To investigate the significance of p53 functionality on sensitization by inhibitors, we stratified 14 samples from the cohort according to ability to upregulate p21 after IR treatment (FACS analysis). 6/10 cases that displayed impaired p21 upregulation (associated with defects in p53 or ATM function) were sensitized to either CLB or fludarabine by NU7441. 4/4 cases with normal p21 response were sensitized by NU7441. These data are key, since they suggest that NU7441 may have efficacy in cases with p53 dysfunction, a group of tumors representing poor prognosis, drug-resistant patients. Current studies are characterising the cohort (11q,17p deletions) and measuring kinetics of DNA DSB formation and repair. Future studies will further investigate effects of targeting ATM in CLL, and targeting DNA-PK in ATM-defective tumors. Use of DNA-PK and ATM inhibitors represents a targeted and novel therapeutic approach for poor prognosis CLL.