Targeting DNA-dependent Protein Kinase Research Articles

Abstract LB-A11: Targeting DNA-PK and the DNA damage response via small molecule Ku inhibitors

Abstract The DNA dependent protein kinase (DNA-PK) is a validated target for cancer therapeutics that drives the DNA damage response (DDR) and plays a critical role as a primary sensor in the non-homologous end joining (NHEJ) DNA double strand break (DSB) repair pathway. Various anti-cancer therapeutic strategies mediate their cytotoxic effects by inducing DSBs, including ionizing radiation (IR), and clinical outcomes are directly related to the repair of DNA damage. Modulating the pathway responsible for repairing DSBs will have a profound impact on the efficacy of DNA damaging agents in the clinic. To date, development of inhibitors for DNA-PK has focused on targeting the active site with ATP mimetics. We have taken the novel and innovative approach to inhibiting DNA-PK via blocking the Ku 70/80 heterodimer interaction with DNA, a necessary step in DNA-PK activation. Exploiting this unique mechanism of kinase activation, we have identified a series of highly potent and specific DNA-PK inhibitors that impart their inhibitory activity via disruption of the binding of Ku protein to DNA ends. This novel approach affords significant advantages to current approaches in kinase inhibition. Novel derivatives of our initial hit inhibit DNA-PK catalytic activity at nanomolar concentrations and potentiate cellular sensitivity to DSB-inducing agents like etoposide and bleomycin. Data demonstrate that the cellular effects observed are a function of Ku inhibition and that this novel class of DNA-PK inhibitors can be further developed as anti-cancer therapeutics that can be used as an adjuvant to, or concomitant with radiotherapy and other cancer therapies that induce DNA damage. Citation Format: John J. Turchi, Navnath Gavande, Pamela S. VanderVere-Carozza, Tyler Vernon, Katherine S. Pawelczak. Targeting DNA-PK and the DNA damage response via small molecule Ku inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A11.

Inhibition of DNA-dependent protein kinase promotes ultrasound-induced cell death including apoptosis in human leukemia cells

Ultrasound (US) has been shown to induce cell death in cancer cells; however, the underlying mechanism remains elusive. Here, we report a set of novel findings on the molecular mechanism. We found that Akt (also known as protein kinase B), a substrate of DNA-dependent protein kinase (DNA-PK), was phosphorylated in U937 cells nullified with p53 or Molt-4 cells artificially abrogated with p53 after US exposure. On the contrary, Akt phosphorylation was transiently down-regulated then recovered in Molt-4 cells harboring wild-type p53 in US-exposed cells, possibly due to a mutual regulation between p53 and Akt. Inhibition of ataxia-telangiectasia mutated (ATM) or DNA-PK revealed that DNA-PK, rather than ATM, was preferentially involved in Akt phosphorylation and cell survival after US-exposure in all cell lines. These results indicate that DNA-PK plays a protective role against US-induced cell death regardless of p53 phenotype. In conclusion, our findings provide the first delineation of the role of DNA-PK in US-induced cell death and suggest that targeting DNA-PK might be a promising strategy to augment cancer eradication by US.

PI3K-like kinases restrain Pim gene expression in endothelial cells

Pim kinases contribute to tumor formation and development of lymphoma, which shows enhanced DNA replication, DNA recombination and repair. Endothelial cells^(ECs) express all the three members of Pim kinase gene family. We hypothesized that DNA repair gene would regulate Pim expression in ECs. Human umbilical vein endothelial cells (HUVECs) were isolated and maintained in M199 culture medium. The cellular distribution of Pim-3 in ECs was determined by immunofluorescent staining. The siRNA fragments were synthesized and transfected by using Lipofectamine LTX. The total cellular RNA was extracted from the cells by using Trizol reagent. cDNAs were quantified by semi-quantity PCR. The effects of LY294002 and wortmannin on RNA stability in ECs were also examined. Our data showed that LY294002 and wortmannin, phosphatidylinositol 3-kinase (PI3K) and PI3K-like kinase inhibitors, increased Pim mRNA expression in ECs without altering the mRNA stability. RNA interference (RNAi) targeting DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) increased mRNA expression of Pim-3 and Pim-1, respectively. Silencing of Akt decreased Pim-1 instead of Pm-2 and Pim-3 gene expression in ECs. But etoposide, a nucleoside analogue, which could activate DNA-PKcs and ATM, increased Pim expression in ECs. Our study indicates that the expression of Pim kinases is physiologically related to DNA-PKcs and ATM in ECs.

DNA-Dependent Protein Kinase Is a Therapeutic Target in Poor Prognosis B-Cell Chronic Lymphocytic Leukemia

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.

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.