DNA-Dependent Protein Kinase Is a Therapeutic Target in Poor Prognosis B-Cell Chronic Lymphocytic Leukemia
Elaine Willmore, BSc, PhD1,
Sarah L. Elliott1,*,
Clark J. Crawford1,*,
Duncan Gowans6,* and
Barbara W. Durkacz1,*
1 Northern Institute for Cancer Research, Newcastle Upon Tyne, United Kingdom, 2 Queen Elizabeth Hospital, Gateshead, United Kingdom, 3 Haematology, Newcastle University, Newcastle upon Tyne, United Kingdom, 4 Royal Victoria Infirmary, Newcastle upon Tyne, United Kingdom, 5 St James University Hospital, Leeds, United Kingdom, 6 Sunderland Royal Hospital, Sunderland
Poor prognosis B-cell chronic lymphocytic leukemia (CLL) ischaracterised by del(17p), del(11q) and unmutated IgVH genes.Mutational inactivation of p53 and ataxia telangiectasia-mutatedkinase (ATM) are more frequent in these patients and conferdrug-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 correlateswith chemo-resistance. Thus, alterations in DNA damage signallingpathways are associated with poor risk CLL. We have shown thatDNA-PK is a new therapeutic target in CLL1, and are evaluatingthe efficacy of novel small molecule inhibitors of DNA-PK inex vivo studies using leukemic lymphocytes from a well-characterisedcohort of CLL patients (n=85). We hypothesised that targetingDNA-PK would inhibit NHEJ and thus sensitise CLL cells to drug-inducedDNA damage.
NU7441 and KU-0060648 are potent small molecule inhibitors ofDNA-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-dependentdecrease in viability in response to single agent treatment(XTT/apoptosis assays) that was potentiated in the presenceof a DNA-PK inhibitor. For example, 14/18 cases tested withmitoxantrone (currently in clinical trials) were sensitisedby NU7441. Measurement of H2AX foci formation (a surrogate markerfor DSB) after Mitoxantrone treatment showed foci formationwithin 3 hr (n=4), which was maximally potentiated at 24hr followingco-incubation with NU7441, implicating DNA-PK as a mediatorof DSB repair following drug treatment. Stratification by karyotypicstatus demonstrated striking results. Although del(17p) caseswere 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 Mitoxantroneby NU7441 (p=0.0006), indicating the particular effectivenessof this combination in del(17p) cases. Consistent with thisobservation, DNA-PK expression (Western blot and activity assays)was highest in del(17p) cases, confirming the utility of thisnovel drug combination. Whereas Topoisomerase II expressionwas negligible (Western blotting), Topoisomerase IIβ expressionvaried 3-fold. RT PCR analyses are underway to further studyexpression of DNA-PK and Topoisomerase II in this cohort. Takentogether, these data show that use of a DNA-PK inhibitor increasesthe therapeutic index of drugs currently used to treat CLL andidentify a targeted and novel approach for poor prognosis disease.
Disclosures: No relevant conflicts of interest to declare.