Role Of DNA-dependent Protein Kinase Research Articles

Abstract 2036: Plasticity and vulnerability associated with extrachromosomal and intrachromosomal BRAF amplifications

Abstract Cancer cells display two modes of focal amplifications (FA): extrachromosomal double minutes (DMs/ecDNAs) and intrachromosomal homogenously staining regions (HSRs). Understanding the plasticity of these two modes is critical for preventing targeted therapy resistance. We developed a combined BRAF plus MEK inhibitor resistance melanoma model that bears high BRAF amplifications through both ecDNA and HSR modes, and investigated FA dynamics in the context of drug resistance plasticity. Cells harboring FAs displayed mode switching between ecDNAs and HSRs, from both de novo genetic changes and selection of pre-existing subpopulations. We found that copy number plasticity is not exclusive to ecDNAs. Single cell-derived clones with HSRs also exhibit BRAF copy number and corresponding HSR length plasticity that allows them to respond to dose reduction and recover from drug addiction. In addition, upon kinase inhibitor escalation, we observed reproducible selection for cells with BRAF kinase domain duplications residing on ecDNAs. Whole genome sequencing of sublines with different FA formats allowed fine reconstructions of BRAF amplicon and revealed conserved structures during the transitions due to drug dose manipulations. The amplification boundaries in our cell line model were found consistent with those in clinically observed cases of combined BRAF/MAPK inhibitor resistance. RNA-seq and CRISPR screening performed on parental, ecDNA and HSR sublines revealed distinct biology and vulnerabilities associated with each kind of FA. Vulnerabilities for genes involved in ecDNA maintenance in DNA repair pathways identified in our model are consistent with previous reports, such as a role for DNA-dependent protein kinase (DNA-PK/PRKDC). Our screen reveals many new targets. In sum, the karyotypic plasticity of FAs allows cancer cells to respond to drug dose challenges through a myriad of mechanisms, including increases or decreases in DMs, shortening of HSRs, and acquisition of secondary resistance mechanisms. The cellular vulnerabilities identified in our study provide roadmaps for therapeutically targeting each kind of FA mode. Citation Format: Kai Song, Jenna Minami, Trevor Ridgley, Arthur Huang, Rachana Jayaraman, William Crosson, Jesus Salazar, Eli Pazol, Senaratne Niroshi, Rao Nagesh, Kim Paraiso, Thomas Graeber. Plasticity and vulnerability associated with extrachromosomal and intrachromosomal BRAF amplifications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2036.

DNA-PK senses DNA virus infection in human cells

Abstract The type I interferon (IFN-I) response to virus infection is initiated by the detection of nucleic acids by intracellular pattern recognition receptors (PRRs). The sensing of DNA viruses by PRRs is carried out by a number of DNA-binding PRRs that signal via the adaptor protein stimulator of interferon genes (STING) to drive IFN-I transcription. We previously described the role of DNA-dependent protein kinase (DNA-PK) as a viral DNA sensor in murine fibroblasts. In this study we show that DNA-PK is essential for the host response to DNA and DNA viruses in human fibroblasts. In the absensce of the catalytic subunit of the DNA-PK heterotrimer, DNA-PKcs, fibroblasts are deficient in their ability to activate a IFN-I response to DNA. DNA-PKcs is activated rapidly following exogenous DNA transfection or DNA virus infection and is required for signalling via STING and the kinase TBK-1 to the transcription factor interferon regulator factor 3 (IRF-3). Most wild-type DNA viruses combat intracellular DNA PRRs using immunomodulatory proteins encoded in their genomes and are effective at blocking IFN-I responses in infected cells. Here we make use of attenutated vaccina and herpes simplex 1 viruses that are lacking the immunomodulators that target DNA sesining mechanisms. We show that DNA-PKcs can sense these viruses and is required for triggering the IFN-I response in infected cells. These data cement the role of DNA-PK in the sensing of DNA virus infections in human cells.

DNA dependent protein kinase (DNA-PK) enhances HIV transcription by promoting RNA polymerase II activity and recruitment of transcription machinery at HIV LTR.

Despite reductions in mortality from the use of highly active antiretroviral therapy (HAART), the presence of latent or transcriptionally silent proviruses prevents HIV cure/eradication. We have previously reported that DNA-dependent protein kinase (DNA-PK) facilitates HIV transcription by interacting with the RNA polymerase II (RNAP II) complex recruited at HIV LTR. In this study, using different cell lines and peripheral blood mononuclear cells (PBMCs) of HIV-infected patients, we found that DNA-PK stimulates HIV transcription at several stages, including initiation, pause-release and elongation. We are reporting for the first time that DNA-PK increases phosphorylation of RNAP II C-terminal domain (CTD) at serine 5 (Ser5) and serine 2 (Ser2) by directly catalyzing phosphorylation and by augmenting the recruitment of the positive transcription elongation factor (P-TEFb) at HIV LTR. Our findings suggest that DNA-PK expedites the establishment of euchromatin structure at HIV LTR. DNA-PK inhibition/knockdown leads to the severe impairment of HIV replication and reactivation of latent HIV provirus. DNA-PK promotes the recruitment of Tripartite motif-containing 28 (TRIM28) at LTR and assists the release of paused RNAP II through TRIM28 phosphorylation. These results provide the mechanisms through which DNA-PK controls the HIV gene expression and, likely, can be extended to cellular gene expression, including during cell malignancy, where the role of DNA-PK has been well-established.

The crucial role of DNA-dependent protein kinase and myelin transcription factor 1-like protein in the miR-141 tumor suppressor network

ABSTRACT Glioblastoma is the most aggressive brain tumor. Although miR-141 has been demonstrated to primarily function as a tumor suppressor in numerous malignancies, including glioblastoma, the mechanisms involved remain poorly understood. Here, it is shown that miR-141 is downregulated in glioblastoma cell lines and tissues and may exert its biological function via directly targeting myelin transcription factor 1-like (MYT1L). Using two glioblastoma cell lines that differ from each other by the functionality of DNA-dependent protein kinase (DNAPK), a functional involvement of DNAPK in the miR-141 tumor suppression network was observed. In M059K cells with a normal function of DNAPK, the enforced expression of miR-141 attenuated MYT1L expression and suppressed cell proliferation. Conversely, the inhibition of miR-141 expression promoted cell proliferation; however, in M059J cells with a loss-of-function DNAPK, miR-141 constitutively inhibited cell proliferation upon ectopic overexpression or inhibition. An overexpression of miR-141 suppressed M059J cell migration, while it had no effect on M059K. Furthermore, the ectopic expression of miR-141 induced an S-phase arrest in both cell lines, whereas the inhibition of miR-141 caused a G1 arrest in M059J and accelerated the S phase in M059K. An overexpression and suppression of miR-141 resulted in an aberrant expression of cell-cycle proteins, including p21. Moreover, MYT1L may be a transcription factor of p21 in p53-mutant cells, whereas DNAPK may function as a repressor of MYT1L. The findings revealed the crucial role of DNAPK in miR-141-mediated suppression of gliomagenesis and demonstrated that it may be a target molecule in miR-141-associated therapeutic interventions for glioblastoma.

The catalytic subunit of DNA-PK has a unique function in inflammation independently of Ku70 and DNA repair: a new opportunity to target the enzyme without interfering with DNA repair

Abstract Our laboratory demonstrated a critical role for DNA-dependent protein kinase (DNA-PK) in asthma pathogenesis via modulating the pertinent immune responses. DNA-PK is DNA repair enzyme composed of a catalytic subunit (DNA-PKcs) and two DNA-binding subunits (Ku70 and Ku80). Human cells express high levels of DNA-PK, surprisingly such high levels do not confer increased ability to repair DNA damage. Here we show that the role of DNA-PK in promoting inflammatory responses is independent of its function in the DNA repair. We examined the effect(s) of partial depletion of Ku70 on Ovalbumin (OVA) induced lung inflammation in mouse model of the disease. Of note, depletion of Ku70 by gene heterozygosity causes deficiency in DNA-PK-dependent DNA repair. Unlike the protective effects provided by DNA-PKcs gene heterozygosity, Ku70 heterozygosity did not alter the OVA-induced eosinophilia, mucus hypersecretion, Th2 cytokines production, or OVA-specific IgE upon OVA challenge. Interestingly, Ku70 heterozygosity enhanced methacholine-induced AHR over that of WT mice. Using a cell culture system, we demonstrate that while IL-4 and TNF-α are potent inducers of DNA-PKcs, such activation did not coincide with any detectable DNA damage or repair responses. Remarkably, while Ku70−/− blocked DNA-PKcs autophosphorylation in response to the DNA damage agent, etoposide, it did not affect the kinase in response to TNFα. Our findings suggest that the mechanism by which DNA-PK functions in inflammation is completely unrelated to its role in DNA repair, thus, unraveling a completely novel function for the kinase. More importantly, this provides a window of opportunity to target DNA-PK in inflammatory diseases without interfering with DNA repair processes.

Abstract LB-264: Preclinical evaluation of DNA-PK as a therapeutic target in prostate cancer

Abstract Prostatic adenocarcinoma (PCa) is dependent on androgen receptor (AR) signaling at all stages of disease, as AR activation induces both cell proliferation and survival. Though organ–confined disease can be treated, the response is transient. Reactivation of AR leads to castration resistant prostate cancer (CRPC), which remains fatal. Previously published studies demonstrate that AR activation promotes tumor cell survival and proliferation through a feed-forward loop involving the DNA repair factor DNA-dependent protein kinase (DNA-PK). Recent data from our lab has highlighted the role of DNA-PK in DNA damage repair as well as transcriptional regulation of pro-metastatic signaling. Strikingly, DNA-PK is the most deregulated kinase in metastatic CRPC. DNAPK is highly elevated and hyperactivated, and is an independent predictor of metastasis and overall survival in patients with high-risk disease. Concordantly, DNAPK suppression attenuated DNA-PK functions in NHEJ and transcriptional regulation. DNA-PK inhibition is sufficient to prevent proliferation in vitro and ex vivo, as well as tumor metastasis in vivo. Combined these findings highlight importance of DNA-PK functions as a transcriptional regulator and a mediator of NHEJ DNA repair and nominate it as a therapeutic target in PCa. Data presented here will interrogate the translational capacity of a specific DNA-PK inhibitor, a dual DNA-PK/TOR kinase inhibitor and a specific TOR kinase inhibitor in CRPC models. Currently, CC-115, a dual kinase inhibitor, is the only DNA-PK inhibitor in Phase1/2 trial in combination with Enzalutamide in PCa. Unbiased transcriptomic analyses were performed and will be used to identify mechanisms of action that lead to suppression of PCa growth in vitro and ex vivo. These findings will provide insight in the effectiveness of DNA-PK inhibitors as a single agent or combinatorial treatments and provide rational for its placement in the correct clinical space. Citation Format: Emanuela Dylgjeri, Jonathan F. Goodwin, Christopher M. McNair, Ayesha A. Shafi, Vishal Kothari, Felix Feng, Dana Rathkop, Karen Knudsen. Preclinical evaluation of DNA-PK as a therapeutic target in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-264. doi:10.1158/1538-7445.AM2017-LB-264

Role of DNA-dependent protein kinase in the HIV-1 replication cycle

Role of DNA-dependent protein kinase in the HIV-1 replication cycle

Role of DNA-dependent protein kinase in the HIV-1 replication cycle

Human immunodeficiency virus type 1 (HIV-1) is among the best-studied viruses, but some aspects of HIV-1 biology remain obscure. The role of cell proteins in virus replication raises especially many questions. One of the proteins is DNA-dependent protein kinase (DNA-PK), which performs crucially important functions in the human body. DNA-PK is known to influence at least two stages in the HIV-1 life cycle, the integration of viral genome in cell DNA and transcription of the integrated provirus. Many details regarding this influence remain unresolved. The review summarizes the known data on the DNA-PK role in the HIV-1 life cycle and its influence on the replication of other members of the family Retroviridae. In the beginning of this review there is a short explanation of the DNA-PK cellular functions that are especially important for understanding its role in the HIV-1 replication.

Dendritic cells induce Th2-mediated airway inflammatory responses to house dust mite via DNA-dependent protein kinase

DNA-dependent protein kinase (DNA-PK) mediates double stranded DNA break repair, V(D)J recombination, and immunoglobulin class switch recombination, as well as innate immune and pro-inflammatory responses. However, there is limited information regarding the role of DNA-PK in adaptive immunity mediated by dendritic cells (DCs), which are the primary antigen-presenting cells in allergic asthma. Here we show that house dust mite induces DNA-PK phosphorylation, which is a marker of DNA-PK activation, in DCs via the generation of intracellular reactive oxygen species. We also demonstrate that pharmacological inhibition of DNA-PK, as well as the specific deletion of DNA-PK in DCs, attenuates the induction of allergic sensitization and Th2 immunity via a mechanism that involves the impaired presentation of mite antigens. Furthermore, pharmacological inhibition of DNA-PK following antigen priming similarly reduces the manifestations of mite-induced airway disease. Collectively, these findings suggest that DNA-PK may be a potential target for treatment of allergic asthma.

Abstract 3329: The DNA-PK inhibitor, NU7441, sensitises cancer cells to microtubule-targeting agents and modulates vincristine efflux in multidrug resistant cells.

Abstract Microtubules are involved in the formation of the mitotic spindle, and correct separation of the sister chromatids at mitosis is essential for maintaining genomic stability. Microtubule-targeting drugs arrest rapidly-dividing cells and are important chemotherapeutic agents, but the development of multidrug-resistance (MDR) is a serious clinical problem. Along with its role in DNA double-strand break repair, DNA-dependent protein kinase (DNA-PK) has also been shown to play an important role in stabilising spindle formation and preventing mitotic catastrophe (Shang et al, Cancer Res. 2010). In light of this novel finding, we hypothesised that inhibition of DNA-PK would sensitise cells to microtubule-targeting agents. Pilot studies showed that inhibition of DNA-PK by the selective inhibitor, NU7441, caused greater sensitisation to vincristine in multidrug-resistant cells compared to parental cells. Here, we further investigate the role of DNA-PK in response to microtubule-targeting drugs using a novel series of NU7441 analogues, and investigate whether these inhibitors interact with the efflux transporter, P-glycoprotein. In growth inhibition studies, CCRF-CEM leukaemia cells were sensitised 1.4-fold to vincristine by NU7441 (1μM) whereas their vincristine-resistant derivative CCRF-CEM VCR/R cells were sensitised 8-fold to vincristine. DNA-PK-deficient M059J glioblastoma cells were 2-fold more sensitive to docetaxel than DNA-PK-proficient cells, and NU7441 sensitised only DNA-PK-proficient M059J-Fus1 cells to docetaxel. CCRF-CEM VCR/R cells have a higher basal level of autophosphorylation of DNA-PKcs (Western blotting) than the CCRF-CEM cells and autophosphorylation in response to vincristine was abrogated by NU7441. To investigate the effects of NU7441 on drug transport, a doxorubicin fluorescence assay showed that in P-glycoprotein-overexpressing canine kidney MDCKII-MDR1 cells, 1μM NU7441 increased doxorubicin cellular fluorescence 16-fold by blocking drug efflux via P-glycoprotein. NU7441 and 3 structurally-related compounds (NU7742 (inactive NU7441 analogue), DRN1 (DNA-PK-inhibitory atropisomeric NU7441 derivative) or DRN2 (DNA-PK non-inhibitory atropisomeric NU7441 derivative)) all increased intracellular vincristine accumulation in the CCRF-CEM VCR/R cells to a level similar to verapamil, as measured by LC-MS. However, the non-inhibitory compound NU7742 was 5-fold less effective at sensitising CCRF-CEM VCR/R cells to vincristine than NU7441 (p=0.008). The novel finding that DNA-PK inhibition sensitises cells to microtubule-targeting agents suggests that along with its function in DNA repair, this enzyme plays a role in the mitotic spindle checkpoint. Additionally, in multidrug-resistant cells, NU7441 is able to block drug efflux via interaction with P-glycoprotein. Citation Format: Emily Mould, Philip Berry, David Jamieson, Christopher Hill, Niu Tan, Sarah Elliott, Barbara Durkacz, David Newell, Elaine Willmore. The DNA-PK inhibitor, NU7441, sensitises cancer cells to microtubule-targeting agents and modulates vincristine efflux in multidrug resistant cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3329. doi:10.1158/1538-7445.AM2013-3329

A role of DNA-dependent protein kinase for the activation of AMP-activated protein kinase in response to glucose deprivation

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) plays an essential role in double-strand break repair by initially recognizing and binding to DNA breaks. Here, we show that DNA-PKcs interacts with the regulatory γ1 subunit of AMP-activated protein kinase (AMPK), a heterotrimeric enzyme that has been proposed to function as a “fuel gauge” to monitor changes in the energy status of cells and is controlled by the upstream kinases LKB1 and Ca2+/calmodulin-dependent kinase kinase (CaMKK). In co-immunoprecipitation analyses, DNA-PKcs and AMPKγ1 interacted physically in DNA-PKcs-proficient M059K cells but not in DNA-PKcs-deficient M059J cells. Glucose deprivation-stimulated phosphorylation of AMPKα on Thr172 and of acetyl-CoA carboxylase (ACC), a downstream target of AMPK, is substantially reduced in M059J cells compared with M059K cells. The inhibition or down-regulation of DNA-PKcs by the DNA-PKcs inhibitors, wortmannin and Nu7441, or by DNA-PKcs siRNA caused a marked reduction in AMPK phosphorylation, AMPK activity, and ACC phosphorylation in response to glucose depletion in M059K, WI38, and IMR90 cells. In addition, DNA–DNA-PKcs−/− mouse embryonic fibroblasts (MEFs) exhibited decreased AMPK activation in response to glucose-free conditions. Furthermore, the knockdown of DNA-PKcs led to the suppression of AMPK (Thr172) phosphorylation in LKB1-deficient HeLa cells under glucose deprivation. Taken together, these findings support the positive regulation of AMPK activation by DNA-PKcs under glucose-deprived conditions in mammalian cells.

Two Cellular Protein Kinases, DNA-PK and PKA, Phosphorylate the Adenoviral L4-33K Protein and Have Opposite Effects on L1 Alternative RNA Splicing

Accumulation of the complex set of alternatively processed mRNA from the adenovirus major late transcription unit (MLTU) is subjected to a temporal regulation involving both changes in poly (A) site choice and alternative 3′ splice site usage. We have previously shown that the adenovirus L4-33K protein functions as an alternative splicing factor involved in activating the shift from L1-52,55K to L1-IIIa mRNA. Here we show that L4-33K specifically associates with the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) in uninfected and adenovirus-infected nuclear extracts. Further, we show that L4-33K is highly phosphorylated by DNA-PK in vitro in a double stranded DNA-independent manner. Importantly, DNA-PK deficient cells show an enhanced production of the L1-IIIa mRNA suggesting an inhibitory role of DNA-PK on the temporal switch in L1 alternative RNA splicing. Moreover, we show that L4-33K also is phosphorylated by protein kinase A (PKA), and that PKA has an enhancer effect on L4-33K-stimulated L1-IIIa splicing. Hence, we demonstrate that these kinases have opposite effects on L4-33K function; DNA-PK as an inhibitor and PKA as an activator of L1-IIIa mRNA splicing. Taken together, this is the first report identifying protein kinases that phosphorylate L4-33K and to suggest novel regulatory roles for DNA-PK and PKA in adenovirus alternative RNA splicing.

Effect of the Inositol Polyphosphate InsP6 on DNA-PK–Dependent Phosphorylation

Inositol hexakisphosphate (InsP(6)) is a member of the inositol polyphosphate group that participates in numerous intracellular signaling pathways. Cheung and colleagues previously reported that InsP(6) stimulated double-strand break repair by nonhomologous end joining (NHEJ) in cell-free extracts and that InsP(6) binding by the Ku70/80 subunit of the DNA-dependent protein kinase (DNA-PK) was required for stimulation of NHEJ in vitro. This report describes InsP(6)-dependent phosphorylation of two NHEJ factors, XRCC4 and XLF, in partially purified human cell extracts. XRCC4 and XLF are known substrates for DNA-PK, which does not require InsP(6) for protein kinase activity. Consistent with a role for DNA-PK in these reactions, InsP(6)-dependent phosphorylation of XRCC4 and XLF was DNA dependent and not observed in the presence of DNA-PK inhibitors. Depletion of the Ku70/80 DNA-, InsP(6)-binding subunit of DNA-PK resulted in loss of InsP(6)-dependent phosphorylation and showed a requirement for Ku70/80 in these reactions. Complementation of Ku70/80-depleted reactions with recombinant wild-type Ku70/80 restored InsP(6)-dependent phosphorylation of XRCC4 and XLF. In contrast, addition of a Ku70/80 mutant with reduced InsP(6) binding failed to restore InsP(6)-dependent phosphorylation. While additional protein kinases may participate in InsP(6)-dependent phosphorylation of XRCC4 and XLF, data presented here describe a clear requirement for DNA-PK in these phosphorylation events. Furthermore, these data suggest that binding of the inositol polyphosphate InsP(6) by Ku70/80 may modulate the substrate specificity of the phosphoinositide-3-kinase-related protein kinase DNA-PK.

2.13 Expression and Function of the NF-κB Subunits in Chronic Lymphocytic Leukemia: A Role for DNA-Dependent Protein Kinase in Regulating DNA Damage–Activated p65 and p50 Subunit Activation

namic gene delivery technique; this permitted hepatocytes to produce and secrete the soluble protein. This technique uses hydrodynamic force, generated by an injection of a large volume (10% of body weight) of DNA solution (30 ig/mL) over 5–7 seconds through the tail vein, to permeabilize the capillary endothelium of the liver and generate ‘pores’ in the plasma membrane of the surrounding parenchymal cells, thereby allowing DNA to reach the cell interior. With time, the membrane pores close, trapping these molecules inside. At that point, transfected cells transiently produce the molecule encoded by the vector. We generated a CD40L construct consisting of the soluble part of human CD40L and the trimerization domain of adiponectin (ADPN-CD40L). We also created a similar construct for human interleukin (IL)-4. The adiponectin domain creates a trimer that can also dimerize to a hexamer, creating multimeric forms with greater biological activity. Furthermore, glycosylation of adiponectin increases the stability of these molecules. After hydrodynamic gene delivery of the ADPN-CD40L or the ADPN-IL-4 expression vector, high levels of ADPN-CD40L ( 5 ng/mL) and ADPN-IL-4 ( 3 ng/mL) were found in the sera of treated mice for more than 6 months. Similar data were obtained when both constructs were injected simultaneously. Next, CFSE-labeled CLL peripheral blood mononuclear cells, followed by anti-CD3 monoclonal antibodies, were injected in these mice. Untreated mice and mice that were co-injected with allogeneic monocytes served as negative and positive controls. Ten days after CLL cell injection into mice with circulating ADPN-CD40L, we documented leukemia cell proliferation in the blood and spleen by CFSE dilution for every CLL sample studied, although the degree of proliferation varied among samples. Nevertheless, proliferation could be extensive; in some cases, leukemic cells underwent more than 6 rounds of division. Mice that received only ADPN-IL-4 did not show enhanced CLL cell proliferation but had increased viability over control animals. Finally, mice that received ADPN-CD40L plus ADPN-IL-4 had the greatest degree of CLL cell proliferation and viability, exceeding that in mice receiving ADPN-CD40L or ADPN-IL-4 alone. At the same time point, no proliferation in untreated mice and negligible or minimal CLL cell proliferation was found in mice receiving monocytes. Thus, this approach reduces or may eliminate a downside of our initial model, in which necessary but uncontrolled T-cell expansion to allo-antigens generated graft-versus-host disease and a graftversus-leukemia effect, making the model short term. This new approach may lead to a long-term model that would allow more complex preclinical studies or would be a better tool to study the basic biology of CLL cells in vivo. Experiments testing this are currently in progress.

2.14 The Kinetics of Phosphorylation of the CD5 Regulatory Domain in Chronic Lymphocytic Leukemia B Cells Reflects Ongoing Activation of the B-Cell Receptor

gene transcription. Similarly, the ataxia telangiectasia mutated kinase (ATM), which senses double strand breaks, is required for DNA damage–induced activation of NFB in breast cancer cells. Furthermore, other reports showed that the DNA damage–signalling enzyme, DNA-dependent protein kinase (DNA-PK) activates NFB by regulating MEK/ERK signalling, the IKK complex, and the p50 subunit. We previously demonstrated that inhibition of DNA-PK chemosensitises CLL cells and that DNA-PK is highly expressed in CLL cells from patients with a poor prognosis. These data collectively led us to hypothesise that, like ATM, part of the DNA-PK–mediated DNA damage response involves regulation of NFB activity. First, we quantified p50, p65, p52, c-rel, and Rel-B subunits in 68 CLL samples (standardised enzyme-linked immunosorbent assay) to determine their role in disease progression and correlation with prognostic markers and resistance. High levels of p50 and p65 subunits correlated with shorter overall survival (hazard ratio 5.8 and 5.7, respectively) and identified patients harbouring del(17p) or del(11q). High levels of p52 and c-Rel had no significant effect on outcome, but in 17 cases of known ATM mutation status, Rel-B levels were significantly higher (p 0.005) in patients with ATM mutations. Increased NFB activity correlated with ex vivo chemoresistance (XTT assay) to fludarabine (p 0.001), chlorambucil (p 0.0001), and mitoxantrone (p 0.03). Of note, in 4 cases, drug-induced activity of the p50 and p65 NFB subunits in fresh CLL cells was abrogated by at least 50% by DNA-PK inhibition (using 1 M of NU7441), indicating that catalytic activity of DNAPKcs is required for drug-induced NFB signalling. In support of this concept, human glioblastoma cells deficient in DNA-PKcs (M059J) were 3-fold more sensitive to the NFB antagonist, BAY 11-7082, than the DNA-PKcs–proficient counterpart (M059J Fus1). These data indicate that in addition to its role in double strand break repair, DNA-PKcs can mediate chemosensitisation by decreasing NFB activity. Pilot data using chromatin immunoprecipitation indicate that NFB subunits are capable of binding to the promoter region of DNA-PKcs, and future studies using knockdown of DNA-PKcs will elucidate the role of DNA-PK on NFB– dependent gene transcription. Collectively, these data suggest that targeting DNA damage activated NFB activity represents a novel approach to overcoming chemoresistance in patients with CLL who have increased levels of the p65 and p50 NFB subunits.

Abstract 2479: Detecting Abelson tyrosine kinase activation by ATM and DNA-PK after exposure to ionizing radiation using a peptide biosensor

Abstract Abelson (Abl) kinase is a non-receptor tyrosine kinase that is involved in many cellular processes, including survival, differentiation and apoptosis. Abl is activated in the response to DNA damage, and is involved in the decision between DNA repair versus apoptosis. It is widely accepted that Abl is regulated in response to DNA double-strand breaks (DSBs) by ataxia telangiectasia mutated protein (ATM) via phosphorylation of Abl at S465. However, other aspects of this complex signaling response have been unclear, including the role of other kinases such as DNA-dependent protein kinase (DNA-PK), because of the difficulties of detecting subtle changes in Abl activation through endogenous substrates. Here, we show Abl is activated in a mutant cell line that cannot be phosphorylated by ATM at S465 (Abl-S465A-EGFP) after exposure to ionizing radiation (IR) using a peptide biosensor we developed for Abl kinase. This biosensor contains the following functional modules: a highly specific Abl kinase substrate, an Abl SH3 binding ligand, a photocleavable linker, a biotin tag and a cell penetrating peptide derived from Hiv-TAT to aid intracellular delivery of cargo across the plasma membrane. Using a combination of chemical biology approaches, Western blot for the phosphorylation of the biosensor peptide and high-resolution MS-based phosphoproteomics, we identified a potential role for DNA-PK in activating Abl kinase after IR, independent of ATM and S465. This information about the importance of DNA-PK in this signaling pathway may lead to a better understanding of c-Abl's function in the response to IR, and could be useful to develop strategies to combat radioresistance or improve radiosensitivity in tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2479. doi:10.1158/1538-7445.AM2011-2479

Relationships Between Aberrant Activity of the NF-κB Subunits and Outcome In Chronic Lymphocytic Leukemia: The Dual Role of DNA Damage Sensor Enzymes

AbstractAbstract 3588Poor prognosis patients with chronic lymphocytic leukaemia (CLL) can be identified by cytogenetic abnormalities such as del(17p), and del(11q) and corresponding mutations in TP53 and ATM (ataxia telangiectasia mutated kinase) respectively, which are associated with chemoresistance by virtue of defects in the DNA damage response pathway. We have demonstrated that overexpression of DNA-dependent protein kinase (DNA-PK), which mediates non-homologous end joining, is also associated with poor prognosis CLL1. Recent data show that constitutive activation of the p65 subunit of the transcription factor, NF-κB, confers poor survival in CLL. Since the repertoire of genes activated by NF-κB includes anti-apoptotic and pro-survival genes, NF-κB therefore represents an attractive target for therapeutic intervention. The parthenolide analogue, LC-1, has been shown to be synergisitic with fludarabine in ex vivo studies on CLL cells2, and has validated the concept of targeting NF-κB and prompted clinical trials in CLL patients. Previous data from our laboratory using cell line models demonstrated that DNA damage-induced NF-κB activation requires the base excision repair protein, poly(ADP-ribose) polymerase (PARP), to confer radioresistance3. The DNA damage sensor, ATM, also mediates the response to DNA damage, via phosphorylation of IKK-γ (Inhibitory κ-Kinase/NEMO). In fact, we demonstrated that radio-sensitization by an ATM inhibitor is mediated via NF-κB, rather than by the inhibition of double strand break repair4. Since these observations identify key DNA damage response proteins as regulators of NF-κB activity, we hypothesized that inhibitors of PARP, ATM and DNA-PK, could chemosensitize CLL cells via inhibition of NF-κB.Here, we analyzed NF-κB activity by quantifying NF-κB subunit DNA binding (measured by ELISA) in an unselected CLL cohort. Constitutive activation of the p65 and p50 subunits correlated closely (P= 0.001, n= 57), and predicted shorter time to first treatment (TTFT) and overall survival (OS). Importantly, higher activation occurred in del(17p) (e.g. p50; P= 0.05, n= 49) cases and in those cases that had received treatment. p52 and c-Rel activation correlated with p65 and p50 activation. However, although high p65 and p50 activation predicted shorter OS and TTFT, increased p52 and c-Rel activation were associated with a longer TTFT (up to 40 months) demonstrating the complex crosstalk between the NF-κB subunits in CLL.We were able to correlate p50 and p65 subunit activation with ex vivo resistance to fludarabine and chlorambucil in 12 cases: the most chemoresistant cases had higher p50 and p65 DNA binding activity. Preliminary data indicates that levels of activated NF-κB binding seen in the ELISA correlates with protein expression in nuclear extracts isolated from CLL cells (P= 0.0013, n=26). Here we make the novel observation that the increase in DNA damage-induced NF-κB activation is linked to increased DNA-PK activation. DNA-PK catalytic subunit levels were significantly higher in patients with high p65 activation (P= 0.02, n= 30), regardless of treatment status. Furthermore, a selective inhibitor of DNA-PK, NU7441, increased mitoxantrone-induced cytotoxicity in CLL cells (up to 50-fold) and reduced p65 and p50 DNA binding, indicating a direct link between DNA-PK and NF-κB activation. Ongoing studies are investigating this mechanistic link further, using chromatin immunoprecipitation (ChIP) and using siRNA knockdown. CLL cells were radiosensitized by either the DNA-PK inhibitor, NU7441 or the pan IKK inhibitor, BAY 11–7082. Strikingly, a combination of these two inhibitors had no further effect on radiosensitization, suggesting that DNA-PK and NF-κB act in a common pathway. We have demonstrated that inhibition of ATM sensitizes CLL cells to DNA damage, and future work will assess the impact of ATM and PARP inhibition on NF-κB activity in CLL cells. These data present a novel role for DNA-PK in the regulation of NF-κB and highlight important new therapeutic avenues for the use of DNA-PK inhibitors, which may prove useful in overcoming NF-κB mediated therapeutic resistance in CLL.Willmore E, et al, Clin Cancer Res. 2008.Hewamana S, et al, Clin Cancer Res. 2008.Hunter JE, et al, Proceedings of the AACR. 2009.Veuger SJ et al, Under review, DNA repair. 2010. Disclosures:No relevant conflicts of interest to declare.

ATM Mutant Chronic Lymphocytic Leukaemia Cells are Chemosensitized by Inhibition of DNA-Dependent Protein Kinase

AbstractAbstract 433Defects in ataxia telangiectasia mutated kinase (ATM) confer poor survival in B-cell chronic lymphocytic leukaemia (CLL). ATM gene deletion (11q23) occurs in approximately 20% of cases, and of these, 38% harbor an ATM mutation on the second allele, resulting in complete loss of ATM function. This prevents signaling of DNA double strand break (DSB) damage and compromises homologous recombinational (HR) repair. Importantly, del(11q) cases without corresponding ATM mutation display wild type ATM activity, with an intact DNA damage response and better outcome than ATM mutated cases1. Thus, in terms of chemotherapeutic resistance, it has been established that not all del(11q) cases should be placed in one group, since those with wild type ATM function have a distinct response. DNA-dependent protein kinase (DNA-PK) is also important for DSB repair and mediates the alternative non homologous end-joining (NHEJ) pathway. We previously showed that increased DNA-PK activity and expression is frequent in poor prognosis CLL and is associated with shorter survival2. We hypothesized that targeting the NHEJ pathway (via DNA-PK inhibition), in ATM deficient CLL cells that already have a defective DNA damage response, would completely compromise the ability of these cells to perform DSB repair, resulting in selective killing of ATM mutant CLL cells.The ATM status of CLL cases was determined using denaturing HPLC followed by confirmatory direct sequencing. Only those cases with pathogenic mutations (e.g. predicted to cause truncations or change in protein structure, or previously reported in A-T patients) were classed as ‘ATM mutant’. The resulting defects in ATM activity in these cases was confirmed by quantifying phosphorylation of downstream ATM targets (e.g autophosphorylation of ATM (ser1981) or phosphorylation of SMC-1) in lysates of ionizing-radiation-treated cells. Ex vivo cytotoxicity using freshly isolated CLL cells was possible in 77 cases of known ATM status. Cells were exposed to increasing concentrations of Fludarabine in combination with the DNA-PK inhibitor, NU7441 (1μ M), for 72 hr, and viability was assessed using an XTT kit. NU7441 potentiated the cytotoxicity of Fludarabine in 31 cases, including 8 cases with a known ATM mutation. As expected, ATM mutant CLL cells were more drug-resistant than those with wild type ATM, but even highly resistant (>20μ M) ATM mutant cases were resensitized up to 20-fold by NU7441. Pilot data using a similar approach showed ATM mutant cases to be 5-fold more resistant to mitoxantrone, but NU7441 potentiated the cytotoxicity of mitoxantrone in ATM mutant cases (n = 3) and ATM wild type cases (n = 5) by up to 6-fold or 3-fold respectively.Preliminary data (n =18) using an ex vivo assay dot blot assay to measure DNA-PK activity correlated DNA-PK activity to expression of the DNA-PK catalytic subunit as determined by Western blotting. Furthermore, DNA-PK activity was significantly higher in ATM mutant, compared to wild type CLL cells (p = 0.008). Current studies are exploring the formation and longevity of γH2AX foci in ATM mutant CLL cells treated with DNA damaging agents, and the role of DNA-PK in repair of DSB in these cells. Also, the link between DNA-PK activity and ATM mutation will be further examined as it provides a possible mechanism and proof of concept of increased sensitization by DNA-PK inhibitors. These results suggest that DNA-PK inhibition can sensitize poor prognosis, ATM mutant CLL cells to chemotherapeutics. Our data are consistent with the concept of synthetic lethality, where tumor cells harboring a DNA repair defect can be killed by targeting the compensatory DNA repair pathway, and suggest a group of patients that may benefit from this combination.1 Austen et al., Journal of Clin. Oncol. 20082 Willmore et al., Clin. Can. Res. 2008 Disclosures:No relevant conflicts of interest to declare.

Involvement of DNA-PK and ATM in Radiation- and Heat-induced DNA Damage Recognition and Apoptotic Cell Death

Exposure to ionizing radiation and hyperthermia results in important biological consequences, e.g. cell death, chromosomal aberrations, mutations, and DNA strand breaks. There is good evidence that the nucleus, specifically cellular DNA, is the principal target for radiation-induced cell lethality. DNA double-strand breaks (DSBs) are considered to be the most serious type of DNA damage induced by ionizing radiation. On the other hand, verifiable mechanisms which can lead to heat-induced cell death are damage to the plasma membrane and/or inactivation of heat-labile proteins caused by protein denaturation and subsequent aggregation. Recently, several reports have suggested that DSBs can be induced after hyperthermia because heat-induced phosphorylated histone H2AX (γ-H2AX) foci formation can be observed in several mammalian cell lines. In mammalian cells, DSBs are repaired primarily through two distinct and complementary mechanisms: non-homologous end joining (NHEJ), and homologous recombination (HR) or homology-directed repair (HDR). DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) are key players in the initiation of DSB repair and phosphorylate and/or activate many substrates, including themselves. These phosphorylated substrates have important roles in the functioning of cell cycle checkpoints and in cell death, as well as in DSB repair. Apoptotic cell death is a crucial cell suicide mechanism during development and in the defense of homeostasis. If DSBs are unrepaired or misrepaired, apoptosis is a very important system which can protect an organism against carcinogenesis. This paper reviews recently obtained results and current topics concerning the role of DNA-PK and ATM in heat- or radiation-induced apoptotic cell death.

Long-Term In vivo Effects of Cisplatin on γ-H2AX Foci Signaling in Peripheral Lymphocytes of Tumor Patients After Irradiation

This study determined the effects of cis-diamminedichloroplatinum(II) on radiation-induced foci formation of gamma-H2AX and Rad51 in lymphocytes. Twenty-eight cancer patients were irradiated for intrathoracic, pelvic, or head and neck tumors and received simultaneous cisplatin containing chemotherapy. The effect of cisplatin on radiation-induced gamma-H2AX and Rad51 foci as a response to ionizing radiation-induced DNA double-strand breaks was measured in lymphocytes after in vivo and in vitro radiochemotherapy. The role of DNA-dependent protein kinase and ataxia-telangiectasia mutated kinase in gamma-H2AX signaling, the consequences of altered gamma-H2AX foci formation on double-strand break end joining, was studied. Cisplatin decreased the number of induced gamma-H2AX foci in lymphocytes after in vivo or in vitro irradiation by 34% +/- 6% at days 0 to 3 after cisplatin (P < 0.0001) and remained significant until day 6. The variation in this cisplatin effect from patient to patient was larger than the retest error within the same patient (P = 0.01). The cisplatin effect was not accompanied by an inhibition of end joining of double-strand break as analyzed using gel electrophoresis of DNA under neutral conditions. Cisplatin also decreased radiation induced Rad51 foci formation in lymphocytes after stimulation of proliferation with phytohemagglutinin by 47% +/- 6% (P < 0.0001). Cisplatin has long-term effects on the early double-strand break response of gamma-H2AX and Rad51 foci formation after ionizing radiation. Inhibition of sensing and processing of double-strand break by gamma-H2AX and Rad51 foci formation are important mechanisms by which cisplatin can alter the radiation response.