DNA-Dependent Protein Kinase Inhibitors As Drug Candidates for the Treatment of Cancer

Molecular Cancer Therapeutics 1257

DNA-dependent protein kinase inhibitors as drug candidates for the treatment of cancer

1 1 Adam Kashishian, Heather Douangpanya, that lesion could be inhibited. As a test of this hypothesis, Darcey Clark,1 Stephen T. Schlachter,2 we asked whether selective inhibitors of DNA-dependent C. Todd Eary,2 Justin G. Schiro,2 protein kinase (DNA-PK) could enhance the toxic effect of Hongmei Huang,2 Larry E. Burgess,2 chemical and physical agents that induce DNA double- Edward A. Kesicki,1 and James Halbrook1 strand breaks (DSBs). DNA-PK participates in DNA DSB repair (DSBR) by 1 ICOS Corporation, Bothell, WA and 2 Array Biopharma, Boulder, CO activating the nonhomologous end-joining pathway (1, 2). DNA-PK is a trimeric protein complex composed of a protein kinase, a DNA-PK catalytic subunit, and a DNA targeting heterodimer Ku70 and Ku80 (3, 4). Cell lines Abstract defective for DNA-PK function (knockout and SCID Cancer presents a difficult challenge for oncologists, as mutant cell lines) are sensitive to killing by DSB-inducing there are few therapies that specifically target disease treatments (5–8). The viridins (9), such as wortmannin and cells. Existing treatment strategies rely heavily on physical the quercitins, are natural product classes, the members of and chemical agents that nonspecifically affect DNA which inhibit both DNA-PK and closely related phospha- metabolism. To improve the effectiveness of these treat- tidylinositol 3-kinases (10). A more potent synthetic ments, we have identified a new class of protein kinase derivative of quercitin, LY294002, also inhibits enzymatic inhibitor that targets a major DNA repair pathway. A phosphorylation of lipids and proteins (11). These moder- representative of this class, 1-(2-hydroxy-4-morpholin- ately potent nonselective kinase inhibitors have cellular 4-yl-phenyl)-ethanone, inhibits the DNA-dependent protein activity,butithasbeendifficulttounambiguously kinase (DNA-PK) and differs significantly from previously distinguish the effects of protein kinase from lipid kinase studied DNA-PK inhibitors both structurally and function- inhibition (12–17). Furthermore, these compounds have ally. DNA-PK participates in the cellular response to and significant cellular toxicity in the absence of induced DNA repair of chromosomal DNA double-strand breaks (DSBs). damage, obscuring the role of DNA-PK in maintaining These new selective inhibitors recapitulate the phenotype DNA integrity. of DNA-PK defective cell lines including those from SCID To develop this concept into a viable cancer therapy, we mice. These compounds directly inhibit the repair of DNA took advantage of our knowledge of DNA-PK defective cell DSBs and consequently enhance the cytotoxicity of lines (5–7, 18–20). Because DNA-PK knockout and SCID physical and chemical agents that induce DSBs but not mutant cell lines are viable, we reasoned that selective other DNA lesions. In contrast to previously studied DNA- small molecule DNA-PK catalytic subunit inhibitors must PK inhibitors, these compounds appear benign, exhibiting sensitize cells to DSB-inducing treatments yet be nontoxic no toxic effects in the absence of DSB-inducing in the absence of DSBs (21–23). Furthermore, these treatments. Most importantly, 1-(2-hydroxy-4-morpholin- compounds must not sensitize cells to other non-DSB- 4-yl-phenyl)-ethanone synergistically enhances radiation- inducing DNA damaging agents. Finally, it was our induced tumor control in a mouse-human xenograft assay. expectation that these compounds must potentiate the These studies validate DNA-PK as a cancer drug target and effect of radiation in an animal tumor model. We find that suggest a new approach for enhancing the effects of these new kinase inhibitors meet these criteria and have existing cancer therapies. (Mol Cancer Ther. 2003;2(12): potential as a new cancer therapy. 1257–1264)

Introduction Materials and Methods Many cancer therapeutics target and disrupt DNA function Cell Lines through adduct formation or by causing single- and Cells were purchased from the American Type Culture double-chain scissions. We speculated that tumor cells Collection (Manassas, VA) and grown as recommended. could be made more sensitive to a specific DNA damaging Culture reagents were from Life Technologies, Inc. agent if the cellular pathway responsible for the repair of (Gaithersburg, MD). Kinase Assays HeLa cell purified (24) DNA-PK was assayed by Received 5/27/03; revised 8/6/03; accepted 9/10/03. measuring phosphorylation of a p53 peptide substrate The costs of publication of this article were defrayed in part by the (EPPLSQEAFADLWKKR). Reaction cocktails contained payment of page charges. This article must therefore be hereby 25-mM HEPES-KOH (pH 7.5), 10-mM MgCl2,0.5-mM marked advertisement in accordance with 18 U.S.C. Section 1734 32 DTT, 50-AM ATP, 0.01-mCi/ml [g- P]ATP, 10-Ag/ml linear solely to indicate this fact. A A Requests for Reprints: James Halbrook, 5812 W. Mercer Way, Mercer plasmid DNA, 200- M p53 peptide substrate, 0.2- g DNA- Island, WA 98046. Phone: (425) 941-5013; E-mail:[email protected] PK (purified from HeLa S3 cells), and test compound.

Reactions were performed at 25jC and terminated after 10 Xenograft Assay min by spotting onto phosphocellulose paper, and radio- BALB/c nude mice (Taconic, Germantown, NY) labeled phosphorylated peptide substrate was measured. engrafted on the right hindquarter with HCT116 cells were Purified lipid kinases were assayed (25) by measuring divided into three groups and treated 4qd Â 2 (days 1 and 32 incorporation of phosphate from [g- P]ATP into phosphoi- 3) when tumors reached a mass of f50 mg. Treatment nositides (Sigma Chemical Co., St. Louis, MO). groups composed of 10 animals each received (a) 4 Â 0.1 ml Colony Forming Assay s.c. injections of formulation vehicle (cremophor EL:PBS, HCT116 cells were incubated with 100-AM IC60211 or 1:3) over 4 h, (b) a single localized dose of 150-rad IC86621 and treated with increasing amounts of g-radiation g-radiation including multiple vehicle-only mock treat- (137Cs Mark I irradiator, J. L. Shepherd & Associates, San ments, or (c) a single localized dose of 150-rad g-radiation Fernando, CA) up to 1600 rad. Alternatively, cells were 30 min after s.c. administration of phospho-IC86621 (0.1 ml) treated with etoposide (Sigma) up to 10 AM and/or IC86621 at 400 mg/kg followed by three more hourly injections at up to 100 AM for 24 h when drugs were removed and cells this dose. Total radiation and drug doses given for the two were incubated in fresh media without drugs. After f1 treatments were 300 rad and 3200 mg/kg, respectively. week, colonies (greater than about 50–100 cells) were fixed, Animals were handled and maintained in strict compliance stained with crystal violet, and scored. with federal guidelines. Tumor mass was determined by Cell Proliferation Assay the empirically derived formula for HCT116 tumor growth (l Â w2)/3.3. Mice were euthanized when a tumor burden Human tumor cell lines were incubated with DNA-PK f inhibitor compounds at varying concentrations and treated of >1000 mg was reached ( 5% of body weight). Log cell with g-radiation up to 800 rad. Cells were grown for 4 kill was determined by the formula log10 (cell kill) = (T À days and then pulsed with 3[H]-thymidine for 12–18 h. C/3.32 Td) (26). Cells were harvested and cellular radioactivity was determined. From these data, the effective concentration Results to achieve 2-fold sensitization (ECTFS) was calculated as Identification of Novel Kinase Inhibitors the drug concentration that enhanced radiation-induced Compounds inhibiting the DNA-PK-dependent phos- cell killing 2-fold at the LD . 90 phorylation of a p53 peptide substrate were identified from DNA DSBR Assay the ICOS Corporation (Bothell, WA) small molecule library. Confluent cultured cells (MDA-MB231, MO59K, and The initial library hit was IC60211 (2-hydroxy-4-morpholin- MO59J) were treated with DNA-PK inhibitor compounds 4-yl-benzaldehyde), an arylmorpholine with an IC50 of 400 or saline for 1 h before chromosomal DNA isolation. Cells nM. Optimization of this moderately potent kinase inhibitor then received (a) no treatment, (b) 25 Gy g-radiation, (c) 25 resulted in many DNA-PK selective inhibitors that main- j Gy and incubated at 37 C for 2 h, or (d) 25 Gy and incubated tained the arylmorpholine substructure (Table 1). Most of j with DNA-PK inhibitor compound at 37 C for 2 h. Cells our studies used IC86621, a methyl ketone derivative of were harvested in ice-cold D-PBS, concentrated, and added IC60211. Although IC86621 was not our most active to warm 2% clean-cut agarose (Bio-Rad, Richmond, CA). compound, it was a convenient, easily synthesized, and Agarose embedded cell slurries were proteinase K treated at chemically stable representative of this inhibitor class. This j 50 C overnight. Deproteinated agarose plugs were cast in compound was also more amenable to in vivo studies 1% low melt agarose gels (Bio-Rad) and DNA was described below. Because IC86621 was structurally very fractionated by pulsed field gel electrophoresis. Chromo- simple, we were surprised to find that this compound had somal DNA was visualized with SYBR Gold (Molecular activity neither against a panel of distantly related protein Probes, Eugene, OR) and quantified with the Storm 860 kinases, including cyclin-dependent kinase 2, Rous sarcoma (Molecular Dynamics, Sunnyvale, CA). tyrosine kinase, aurora-related kinase 2, protein kinase A, DNA Damage-Induced Replication Protein A protein kinase C, checkpoint protein kinase 1, and casein Phosphorylation kinase 1, nor against the closely related protein kinases, HCT116 were serum starved (0.5% fetal bovine serum) including ataxia telangiectasia mutant protein kinase, ataxia for 24 h and treated with 2-mM hydroxyurea (Sigma) for telangiectasia mutant/Rad3-related kinase, and FKBP12/ 18 h. Synchronized cells were treated with 16-AM campto- rapamycin-associated protein kinase (at up to 100 AM; data thecin (Sigma) for 2 h with or without varying amounts of not shown; 27–29). IC86621 and related compounds do IC86621. Cells were lysed in the presence of protease have activity against the closely related phosphatidylinosi- inhibitors, fractionated by SDS-PAGE, and transferred to tol 3-kinases (Table 1). However, IC86621 and IC486154 are Immobilon (Millipore, Bedford, MA). Protein filters were selective with respect to phosphatidylinositol 3-kinase probed with anti-replication protein A (RPA; Tom J. Kelly, catalytic subunit (p110) a, g, and y but are equipotent with Johns Hopkins University, Baltimore, MD) and anti-h-actin p110h. The more highly evolved morpholino-flavonoid, (Oncogene Science, San Diego, CA) followed by HRP-GaM IC87361, is 50-fold more selective for DNA-PK than for (Bio-Rad). Immunoblots were visualized with enhanced p110h. Despite the crossover into the lipid kinase family, chemiluminescence (Pierce Chemical Co., Rockford, IL). none of these compounds were cytotoxic at concentrations Protein band intensity was quantified using the Storm 860 of <50 AM as determined by measuring trypan blue dye densitometer (Molecular Dynamics). uptake and 3[H]-thymidine incorporation (data not shown).

Table 1. Activity of representative DNA-PK inhibitors

a Compound Structure IC50 (nM) IR sensitization DNA DSBR b c ECTFS (AM) EC50 (AM) DNA-PK p110a p110h p110g p110y

IC86621 120 1400 135 880 1000 50 68

IC486154 44 890 42 490 180 29 29

IC87102 35 2700 400 1800 5000 20 25

IC87361 34 3800 1700 2800 7900 7 8

Wortmannin 150 38 45 14 70 >50 25

LY294002 360 1000 920 600 4000 >50 50 aLipid kinase activity was determined by averaging the results of two independent assays. bIonizing radiation sensitivity was determined by averaging the results from at least three cell proliferation assays. cInhibition of DNA DSBR was determined from the average of two dose response curves.

DNA-PK Inhibitors Recapitulate the SCID Phenotype IC86621 Specifically Affects the Cellular Metabolism To support continued drug development of these lead of DNA DSBs compounds, it was necessary to demonstrate proof of We tested a broad panel of chemotherapy drugs to see concept at the cellular level. We first asked whether these if IC86621 enhanced cell killing with any of these agents. compounds sensitized cells to DSB-inducing agents. IC86621 did not enhance tumor cell killing by vinblas- IC60211 and IC86621 both enhance radiation-induced cell tine, doxorubicin, paclitaxel, cisplatin, cyclophosphamide, killing (Fig. 1A). At a fixed drug concentration, IC86621 is 5-fluorouracil, cytosine arabinoside, 6-mercaptoguanine, more potent than IC60211, reflecting its lower IC50 (see methotrexate, or camptothecin (data not shown). How- above). Radiation-induced cell killing was enhanced f4- ever, IC86621 significantly enhanced the cellular toxicity fold by coincubation with IC86621 at the LD90. Radiation of etoposide and bleomycin, two agents that induce DSBs enhancement was also measured by comparing terminal (31–33). We examined drug-dependent potentiation of survival slopes using the metric D 0 (radiation dose etoposide-induced cell killing in more detail (Fig. 1B). We required to reduce survival 1/e; 30). IC86621 reduces D0 found that at 100-AM IC86621 etoposide-induced tumor 3-fold from 150 to 50 rad. This is in good agreement with cell toxicity was enhanced 15-fold. Therefore, only repair D0 values published for cells cultured from SCID mice. of DNA DSBs but not single-strand breaks, alkylation, or BALB/3T3 fibroblasts were found to be 2.9-fold more other DNA adducts was blocked by these selective DNA- resistant than SCID fibroblasts (257 vs. 89 rad; 7). In a PK inhibitors. Furthermore, these inhibitors failed to similar experiment, granulocyte/macrophage-colony form- enhance cell killing by the non-DNA damaging agents, ing unit from wild-type mice were 2.4-fold more resistant paclitaxel and vinblastine. than the comparable cells from SCID mice (155 vs. 65 rad; 5). We confirmed the mechanism by which our DNA-PK Cells cultured in the presence of up to 50-AM IC86621 inhibitors enhanced cell killing by directly examining the showed no decrease in cell viability. At drug levels greater effect on chromosomal DNA DSBR (Fig. 2, A and B). Three than 200 AM, IC86621 cell growth rate was retarded but cells different arylmorpholines, IC86621, IC486154, and IC87102, remained viable during 2 weeks of continuous culture in and wortmannin blocked religation of radiation-induced the presence of drug (data not shown). This effect was chromosomal DNA DSBs in a dose-dependent manner. We reversible as a normal growth rate was reestablished when also examined chromosomal DNA DSBR in the DNA-PK drug was removed from the culture medium. normal and defective cell lines MO59K and MO59J,

of the total cellular protein pool to observe a significant effect. Therefore, we find the discrepancy between IC50 and EC50 values to be consistent with our understanding of DNA-PK function. We also asked whether IC86621 could block intracellular DNA-PK-dependent protein phosphorylation. Camp- tothecin treatment of S-phase-arrested cells induces a DNA-PK-dependent phosphorylation of the RPA (35). We corroborated and extended this observation by demon- strating that IC86621 inhibited RPA phosphorylation under these conditions in a dose-dependent manner (Fig. 3). Moreover, the drug concentration required to achieve this effect was similar to that required to inhibit chromosomal DNA DSBR. Our observations support the hypothesis that blocking DNA-PK activity inhibits DNA DSBR and enhances the cellular toxicity of DNA DSB- inducing treatments. DNA-PK Inhibitors Enhance Radiation-Induced Tumor Control Our expectation was that these inhibitors would block DNA DSBR independent of cell type. Human tumor cells of varied origin and genetic background were screened for drug-dependent radiation sensitivity. We found that all cell lines tested exhibited similar sensitivity to IC86621 ranging from 1.5- to 4.2-fold (Table 2). We do not believe these differences to be significant but rather that all cell lines are likely to be sensitive to this approach. We therefore selected the HCT116 cell line for xenograft testing as this cell line grows rapidly in culture, is easily engrafted into nude mice, and has a moderately rapid and reproducible tumor doubling time as a xenograft. To demonstrate in vivo proof of concept, we chose to Figure 1. DNA-PK inhibitors enhance DNA DSB-induced cellular examine drug-dependent potentiation of radiation-induced toxicity. A, cell viability following ionizing radiation without drug (.), or with 100-AM IC60211 (n) or IC86621 (E). B, cell viability following tumor cell killing instead of etoposide- or bleomycin- etoposide treatment without drug (.), or with 25AM (n), 50AM (E), or induced tumor cell killing because of the ease of radiation x 100AM ( ) IC86621. Cell viability was determined by colony forming administration and for pharmacokinetic simplicity. With assay (averaged from two experiments). radiation as the DSB-inducing agent, we had only to control IC86621 drug levels. Furthermore, we did not have potential concerns regarding drug-drug interactions that respectively (Fig. 2C). We corroborated previous observa- might alter compound metabolism and elimination. De- tions that DNA DSBR is severely crippled in MO59J but not tailed cellular studies characterizing IC86621-dependent MO59K cells, suggesting that this DNA repair reaction is radiation-induced cell killing indicated that maximal DNA-PK dependent (34). These data directly demonstrate activity was observed when cells were exposed to 50-AM that our selective DNA-PK inhibitors enhance radiation- drug for 10 h following radiation (data not shown). induced tumor cell killing as a consequence of disrupting However, because of solubility limitations and rapid DNA DSBR. We tested over 35 inhibitor compounds for clearance of IC86621 from plasma, we were unable to inhibition of DNA DSBR and found a similar strong maintain these levels in mice. We partially overcame these correlation among DNA-PK inhibitory activity, radiation obstacles by administration of a phosphorylated prodrug of sensitivity, and chromosomal DNA DSBR activity (data IC86621. Addition of phosphate to the free phenolic group not shown). greatly increased solubility, which permitted us to main-

The EC50 values for these inhibitor compounds are tain drug levels of >50 AM for 4 h. Following administration significantly greater than their IC50 values (Table 1). This of phospho-IC86621, we found only parent compound in property is not uncommon among protein kinase inhibitors plasma as the phosphoester bond is very labile in vivo. and may be due to several factors including cellular Although 4-h drug exposure was less than our cell-based penetration and competition with endogenous ATP levels. efficacy studies suggested, we were keen to test our DNA-PK is a highly abundant cellular protein that likely hypothesis with this dosing strategy. acts stoichiometrically in the end-joining process. Conse- Nude mice engrafted with HCT116 human colon quently, it may be necessary to inhibit a very large fraction carcinoma cells were divided into three treatment arms

Figure 2. DNA-PK inhibitors block the repair of chromosomal DNA DSBs induced by ionizing radiation. A, fractionation of chromosomal DNA from MDA- MB231 cells by pulsed field gel electrophoresis. Following ionizing radiation, cells were allowed to repair damage for 2 hrs with or without increasing amounts of IC86621. DNA extracts were analyzed in duplicate. B, graphical representation of data. DNA damage index, the ratio of broken DNA to intact DNA. C, fractionation of chromosomal DNA from MO59K and MO59J cell lines. Top and bottom DNA staining band, intact, undamaged chromosomes and broken, damaged chromosomes, respectively.

when tumors reached a mass of f50 mg. Because drug availability was limiting, we were unable to include a drug-only treatment group. However, previous xenograft studies with this compound indicated that drug treatment without radiation exerted no effect on tumor growth rate consistent with our observations from cellular studies. To optimally use available drug, we performed two cycles of drug and radiation treatments on days 1 and 3. We found that IC86621 exerted a profound enhancement of radiation-induced tumor control (Fig. 4A). Whereas radiation treatment (300 rad) extended the median time of tumor growth to 1000 mg from 25 to 29 days, drug combined with these two radiation treatments extended the median time to 45 days. This represents a 4-fold increase (4 vs. 16 days) in the effectiveness of radiation under these conditions. The delay in tumor growth rate also translated into a 4-fold survival benefit (10% vs. 40%) at 45 days and a 2-fold Figure 3. IC86621 blocks intracellular DNA-PK-dependent phosphory- survival enhancement when the experiment was con- lation of RPA following DNA damage. HCT116 cells synchronized in S-phase were treated with camptothecin to induce DNA DSBs and cluded on day 55 (Fig. 4B). Similar results were also increasing amounts of IC86621. The average of two experiments is observed in a second experiment and in xenografts using reported.

Table 2. Drug-induced radiation enhancement is cell type We find that maximal radiation enhanced tumor cell independent killing is 2–4-fold in vitro. Similar enhancement was observed in the xenograft assay when measured by either Cell line Cell type Radiation enhancement tumor growth delay or survival (time to 1000 mg). Estimating the degree of tumor cell killing (26) in vivo HCT116 Human colon carcinoma 2.9 from the data presented in Fig. 4A, we calculated log kill SW620 Human colon adenocarcinoma 2.4 to be 1.8. This translates to tumor cell killing of 65-fold. HL60 Human premyelocytic leukemia 4.2 This significant tumor activity was achieved despite MOLT4 Human lymphoblast 2.1 reaching only 40% of our target drug exposure level. H322 Human bronchioalveolar carcinoma 1.6 Because the dosing regimen can be optimized further and SKOV-3 Human ovary adenocarcinoma 1.9 radiation treatments can be increased to more closely OVCAR-3 Human ovary adenocarcinoma 2.4 simulate a clinical treatment course, we believe that PC-3 Human prostate adenocarcinoma 1.5 radiation-induced tumor control beyond what we have

Radiation enhancement is the ratio of the LD90 radiation dose (without drug) to the LD90 radiation dose (100-AM IC86621) from proliferation assay (averaged from two experiments).

tumors engrafted from the human breast carcinoma cell line, MDA-MB231 (data not shown). These data demonstrate that chemical inhibition of DNA-PK enhances radiation-induced tumor control.

Discussion DNA-PK represents a new target for cancer drug development. As a target for radiation enhancement, this approach differs significantly from previous attempts to develop radiation sensitizers (36). In contrast to oximimetic and bioreductive radiosensitizers that have failed clinically, these DNA-PK inhibitors appear benign and possess no inherent antitumor or cytotoxic activity. This attractive property suggests that combining enhancer drug with radiation or chemical DNA DSB-inducing chemotherapy will not add confounding toxicities to a clinical treatment strategy. The synergistic radiation enhancement we observe is unprecedented for a drug with no activity as a single agent. The activity of these inhibitors is consistent with observations previously reported for DNA-PK defective cell lines (5, 7). We find that these inhibitors enhance the toxicity of chemical and physical agents that induce DNA DSBs but not other DNA lesions. Interestingly, the DNA- PK inhibitors do not enhance the cytotoxicity of camptothecin in culture. Camptothecin induces DNA single-strand breaks that are converted to DSBs during DNA replication in S phase (37). Replication-induced DNA DSBs appear to be repaired predominately by homologous recombination and to a lesser extent by the DNA-PK-dependent nonhomologous end-joining pathway (38). Cell cycle responses to camptothecin appear unaffected in MO59J cells, suggesting that signaling by DNA-PK may play an important albeit noncritical role in camptothecin-induced DNA damage (39). Although DNA-PK-dependent phosphorylation of Figure 4. IC86621 enhances radiation-induced tumor control in a DNA metabolic proteins is activated by camptothecin (34, mouse-human xenograft cancer model. Treated animals received radiation 40), our observations support the hypothesis that the repair (150 rad) and drug (1600 mg/kg) on days 1 and 3. A, median (n = 10) of of DNA DSBs generated at replication forks is mediated by tumor masses from untreated control arm (.), radiation-only arm (E), and radiation + drug treatment arm (n). B, Kaplan-Meyer survival plot for the a DNA-PK-independent pathway, perhaps involving ho- untreated control arm (.), radiation-only arm (E), radiation + drug mologous recombination. treatment arm (n). P = 0.414.

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