SummaryDefects in the DNA damage response pathway [e.g. del(17p)] are associated with drug-resistant B-cell chronic lymphocytic leukaemia (CLL). We previously demonstrated that over-expression of DNA-dependent protein kinase (DNA-PK) correlates with chemo-resistance and that inhibition of DNA-PK sensitizes CLL cells to chemotherapeutics. Here, we investigated expression of DNA-PK and other proteins that impact on drug resistance, and evaluated the effects of a DNA-PK inhibitor (NU7441) on mitoxantroneinduced cytotoxicity in CLL cells. NU7441 sensitized cells from 42/49 CLL samples to mitoxantrone, with sensitization ranging from 2-to 200-fold Co-culture of CLL cells in conditioned stromal medium increased chemoresistance but did not reduce sensitization by NU7441. Mitoxantrone treatment induced cH2AX foci and NU7441 increased their longevity (24 h). NU7441 prevented mitoxantrone-induced autophosphorylation of the DNA-PK catalytic subunit (DNA-PKcs) at Ser 2056, confirming that DNA-PK participates in repair of mitoxantrone-induced DNA damage. del(17p) cases were more resistant to mitoxantrone than del(13q) cases, but were resensitized (7-16 fold) by co-incubation with NU7441. Expression of DNA-PKcs, Ku80, P-glycoprotein and topoisomerase IIb were significantly higher in del(17p) cases. PRKDC mRNA levels correlated with DNA-PKcs protein expression, which predicted shorter survival. These data confirm the potential of DNA-PK as a therapeutic target in poor prognosis CLL.

Chronic Lymphocytic Leukaemia (CLL) is the most common adult leukaemia in the western world. It is a highly heterogenic disease with variable disease progression. One of the clearest indicators of poor prognosis in CLL is loss of p53 function via deletion of chromosome 17p13 (del17p) and mutation of TP53. Since TP53 mutation rates rise dramatically in treatment-refractory CLL, identifying TP53 mutations at diagnosis may aid treatment decisions. We have compared next generation (NGS) to Sanger sequencing to determine the optimal method and detection level for TP53 mutation in CLL and assess the clinical significance of small TP53-mutated subclones. Since the incidence of TP53 mutation is relatively low (approximately 10%) when first treatment of CLL is required, targeting the p53 pathway may present a treatment option. To investigate this possibility, primary CLL cells were exposed ex vivo to the MDM2-p53 antagonists Nut-3a, RG-7112 and RG-7388 which prevent the interaction between p53 and its inhibitor protein MDM2 to activate p53 leading to growth inhibition and cell death. We analysed genomic DNA from the CLL cells of 140 patients, including 36 with multiple samples from different stages of disease progression (median follow-up 10 years), by NGS using a ROCHE 454 GS-FLX pyrosequencing platform. In addition, 90 samples were previously screened for TP53 mutations by Sanger DNA sequencing. Full clinical diagnostic and outcome data was available. To assess the functional status of the MDM2-p53 pathway, the sensitivity of CLL cells to a range of non-genotoxic MDM2-p53 antagonists was evaluated. Kaplan Meier analysis of NGS data showed that patients with TP53 mutation+del17p had a median overall survival (OS) of 50 months compared to 140 months for wild type TP53 patients (HR = 2.8, CI 1.7-14.6; p = 0.003). 5% of patients harboured a monoallelic TP53 mutation in the absence of del17p, 2.1% had del17p without mutation on the remaining allele, and 9.3% had del17p+TP53 mutation. Interestingly, patients with del17p+TP53 mutation showed poorer survival than patients with TP53 mutation alone (HR 7.2, CI 1.2-14.6; p = 0.02). One patient with TP53 mutation alone has stable disease (10 years, no treatment) since detection of mutation. Four patients had TP53 mutant samples detected by NGS which were not detected by Sanger sequencing. In addition TP53 WT CLL cells showed a cytotoxic response to MDM2-p53 antagonists (RG-7388 LC50 0.4±0.08 μM, n = 11) whereas TP53 mutant CLL cells were 11 fold less sensitive (RG-7388 LC50 4.58±1.36 μM, n = 3) as determined by XTT assay following 48 hour treatment. NGS is a more sensitive method for detection of TP53 mutations compared with Sanger sequencing. Patients with TP53 mutation alone in the absence of del17p display longer OS compared to patients with del17p+TP53 mutation. In addition, MDM2-p53 antagonists provide a potential future treatment option in TP53 WT CLL. Citation Format: Laura Woodhouse, Clark Crawford, Scott Marshall, Nick Bown, Jonathan Wallis, Geoffrey Summerfield, Elaine Willmore, John Lunec. Assessment and targeting of the MDM2-p53 network in CLL. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5263. doi:10.1158/1538-7445.AM2015-5263

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.