"Schedule-Dependent Synergy between the Heat Shock Protein 90"
Cancer Therapy: Preclinical Schedule-Dependent Synergy between the Heat Shock Protein 90 Inhibitor 17-(Dimethylaminoethylamino)-17-Demethoxygeldanamycin and Doxorubicin Restores Apoptosis to p53-Mutant Lymphoma Cell Lines Ana I. Robles,1 Mollie H.Wright,1 Bheru Gandhi,1 Steven S. Feis,1 Christin L. Hanigan,1 Adrian Wiestner,2 and LyubaVarticovski1 Abstract Purpose: Loss of p53 function impairs apoptosis induced by DNA-damaging agents used for cancer therapy. Here, we examined the effect of the heat shock protein 90 (HSP90) inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG) on doxorubicin-induced apoptosis in lymphoma. We aimed to establish the optimal schedule for administration of both drugs in combination and the molecular basis for their interaction. Experimental Design: Isogenic lymphoblastoid and nonisogenic lymphoma cell lines differing in p53 status were exposed to each drug or combination. Drug effects were examined using Annexin V, active caspase-3, cell cycle, and cytotoxicity assays. Synergy was evaluated by median effect/combination index. Protein expression and kinase inhibition provided insight into the molecular mechanisms of drug interaction. Results: Presence of mutant p53 conferred increased survival to single agents. Nevertheless, DMAG showed synergistic toxicity with doxorubicin independently of p53 status. Synergy required exposure to doxorubicin before DMAG. DMAG-mediated down-regulation of CHK1, a known HSP90 client, forced doxorubicin-treated cells into premature mitosis followed by apopto- sis. A CHK1 inhibitor, SB-218078, reproduced the effect of DMAG. Administration of DMAG before doxorubicin resulted in G1-S arrest and protection from apoptosis, leading to additive or antagonistic interactions that were exacerbated by p53 mutation. Conclusions: Administration of DMAG to doxorubicin-primed cells induced premature mitosis and had a synergistic effect on apoptosis regardless of p53 status. These observations provide a rationale for prospective clinical trials and stress the need to consider schedule of exposure as a critical determinant of the overall response when DMAG is combined with chemotherapeutic agents for the treatment of patients with relapsed/refractory disease. The DNA damage response pathway is a surveillance network telangiectasia mutated (ATM) and ATM and Rad3-related that halts the cell cycle in the presence of damaged DNA to (ATR), which in turn phosphorylate and activate effector prevent genomic instability (1). This signal transduction kinases, such as the serine-threonine kinases CHK1 and CHK2. network is composed of DNA damage sensors that recruit A complex cascade of phosphorylation events further activates phosphatidylinositol 3-kinase – like proteins, including ataxia proteins involved in cell cycle control, DNA repair, and trans- criptional regulation (1, 2). As a result, the cell cycle is delayed and DNA repair or apoptosis ensues. A key downstream target of DNA damage checkpoint activation is the p53 transcription Authors’ Affiliations: 1Laboratory of Human Carcinogenesis, Center for Cancer factor. TP53 is a tumor suppressor gene that is functionally Research, National Cancer Institute and 2Hematology Branch, National Heart, inactivated in more than half of human cancers through gene Lung and Blood Institute, NIH, Bethesda, Maryland Received 5/15/06; revised 8/11/06; accepted 8/24/06. deletion or mutation (3, 4). The p53 protein is normally Grant support: Intramural Research Program of the NIH, National Cancer Institute, expressed at low levels, but on exogenous (radiation, chemo- Center for Cancer Research. therapeutic agents) or endogenous (free radicals) DNA damage, The costs of publication of this article were defrayed in part by the payment of page it becomes stabilized and activated as a transcription factor. charges. This article must therefore be hereby marked advertisement in accordance Depending on cellular context and the level of DNA damage, with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research p53 activation leads to sustained cell cycle arrest, senescence, Online (http://clincancerres.aacrjournals.org/). and/or apoptosis (5, 6). Cells that express wild-type (WT) p53 Requests for reprints: Ana I. Robles, Laboratory of Human Carcinogenesis, undergo G1-S and G2-M arrest after DNA damage. Cells that National Cancer Institute/NIH, MSC 4258, Room 3060, Building 37, 37 Convent express mutant p53 or are p53-null bypass G1-S arrest and must Drive, Bethesda, MD 20892. Phone: 301-496-1729; Fax: 301-496-0497; E-mail: roblesa@ mail.nih.gov. rely on the G2-M checkpoint to undergo DNA repair (2). It has F 2006 American Association for Cancer Research. been proposed that the transient arrest in G2-M is essential for doi:10.1158/1078-0432.CCR-06-1178 recovery from genotoxic stress and that its abrogation may www.aacrjournals.org 6547 Clin Cancer Res 2006;12(21) November 1, 2006 Cancer Therapy: Preclinical result in sensitization of p53-deficient tumor cells to DNA- It has been proposed that the best way to exploit the novel damaging agents and radiation (7, 8). In fact, studies of drug mechanism of action of HSP90 inhibitors is by combination combinations using the protein kinase inhibitor UCN-01 with conventional cytotoxic drugs or other molecularly targeted (7-hydroxystaurosporine, NSC 638850) have shown that agents (14). We found that the schedule of drug administration exposure to UCN-01 increases chemotherapy-induced apopto- determines the efficacy of HSP90 inhibitors when combined sis in cells with disrupted p53 function through abrogation of with the DNA-damaging agent doxorubicin. Because clinical the G2 checkpoint (9, 10). Although UCN-01 was originally trials will likely include patients with relapsed/refractory identified as a protein kinase C inhibitor, it also blocks CHK1 disease, many of whom will bear mutations in TP53, we activity, and this is thought to result in loss of G2 checkpoint investigated how the lack of functional p53 affects the response. control and increased cell death of p53-defective cells (8). We found that clinically achievable concentrations of DMAG Clinical trials are currently under way using UCN-01 alone and abrogated CHK1 expression and doxorubicin-induced G2-M in combination with other agents (11, 12). arrest. Addition of DMAG to doxorubicin-primed cells had a Heat shock protein 90 (HSP90) is a major molecular synergistic effect on survival, regardless of p53 status, and chaperone involved in the conformational folding of many sensitized p53-deficient cells to doxorubicin. cellular proteins, collectively referred to as ‘‘client proteins’’ (13). HSP90 client proteins include a wide variety of signal- transducing regulators of cell growth and differentiation. The Materials and Methods binding of HSP90 to client proteins in a multiprotein chaperone complex is ATP dependent and serves a dual function. In the Cell lines. TK6, NH32, and WTK1 are EBV-immortalized human presence of ATP, HSP90 aids proper folding and stabilizes lymphoblastoid cell lines, with a population doubling time of 14 to 18 proteins, whereas, in its absence, unfolded client proteins are hours (30). TK6 and WTK1 were provided by Dr. Howard Liber targeted for degradation through the proteasome pathway (Colorado State University, Fort Collins, CO) and NH32 was provided (14). HSP90 inhibitors, the naturally occurring ansamycin by Dr. E. Chuang (National Cancer Institute). BJAB, SUDHL-4, OCI- antibiotic geldanamycin (NSC 122750) and its analogues, Ly3, and OCI-Ly10 are non-Hodgkin’s lymphoma cell lines. OCI-Ly3 17-AAG (17-allylamino-17-demethoxygeldanamycin, NSC and OCI-Ly10 are ABC-subtype diffuse large B-cell lymphoma cell lines (WT TP53) and were provided by Dr. Louis Staudt (National Cancer 330507) and DMAG (17-(dimethylaminoethylamino)-17- Institute, Bethesda, MD). BJAB is a Burkitt-like lymphoma cell line that demethoxygeldanamycin, NSC 707545), block the ATP-binding carries a His193Arg mutation in TP53 (31). It has a population pocket of HSP90 and inhibit the essential ATPase activity, doubling time of f36 hours (data not shown). We found no record of leading to destabilization and eventual degradation of HSP90 TP53 status of the diffuse histiocytic lymphoma cell line SUDHL-4. All client proteins. It is becoming apparent that inhibition of cell lines were maintained at 5 Â 105 to 10 Â 105 cells/mL in RPMI HSP90 might be a viable option for the treatment of cancer (15). 1640, supplemented with 10% fetal bovine serum, glutamine, and 1% Binding of HSP90 to 17-AAG is 10- to 100-fold higher in cancer penicillin-streptomycin in a humidified 37jC incubator with 5% CO2. cells than in normal cells. Moreover, cancer cells seem to be Drugs. Doxorubicin was obtained from Sigma (St. Louis, MO) and particularly dependent on growth-promoting and survival dissolved in PBS as 10 mmol/L stock aliquots that were frozen and signal transduction pathways that are affected by the inhibition diluted in media immediately before use. The HSP90 inhibitor geldanamycin and its analogues, 17-AAG and DMAG, were obtained of HSP90. This is exemplified by the sensitization of BCR/ABL – from Cancer Therapy Evaluation Program under Material Transfer positive leukemia cells through HSP90 inhibition (16, 17). Agreement with Kosan Biosciences. The indolocarbazole derivative Other potential tumor targets include those driven by so-called SB-218078 (Calbiochem, San Diego, CA) acts as a potent inhibitor of ‘‘oncogene addiction,’’ a term that describes tumor cells that are CHK1 (32). HSP90 inhibitors and SB-218078 were dissolved in DMSO more sensitive to blockage of constitutively active growth- as 10 mmol/L stock aliquots that were frozen and diluted in media promoting pathways than normal cells. From a broader immediately before use. perspective, down-regulation of multiple signal-transducing Growth inhibition assay. Cells were seeded at 20,000 per well in pathways of cell growth and differentiation by HSP90 inhibitors 96-well plates. Serial dilutions of doxorubicin and/or HSP90 inhibitors may provide an advantage over single-target agents for treat- in medium were added by sextuplicate. Dose-response curves to single ment of tumors bearing multiple oncogenic mutations (18). drugs at 24 and 48 hours after exposure were generated to determine the range of concentrations to be used in combination. For drug 17-AAG and DMAG are effective, alone or in combination with interaction studies, drugs were sequentially added (with the second other agents, in preclinical models of cancer (19 – 21) and are compound introduced with a 24-hour delay) in a final volume of currently in phase I/II clinical trials (22 – 25). Because HSP90 100 AL. Cytotoxicity was measured using the Cell Titer 96 Aqueous One inhibitors have the potential to affect many cellular pathways, Solution Cell Proliferation Assay (Promega, Madison, WI), a colori- the mechanism by which they exert antitumor activity is metric method for determining the number of viable cells based on complex and not well understood. bioreduction of a tetrazolium compound [3-(4,5-dimethyl-thiazol-2yl)- Doxorubicin is a topoisomerase inhibitor that intercalates 5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H -tetrazolium into DNA to induce DNA damage and oxidative stress, which (MTS)] by metabolically active cells. After 24 or 48 hours of exposure to result in cell cycle arrest and apoptosis (26). Treatment for a single drug or after a total of 48 hours in sequential addition experiments, 20 AL of MTS reagent were added to each well and the patients with non-Hodgkin’s lymphoma is based on doxoru- plates were incubated in a humidified 37jC incubator with 5% CO2 for bicin-containing combination chemotherapy, of which cyclo- 1 to 4 hours. Absorbance at 490 nm was recorded using a 96-well plate phosphamide-Adriamycin-vincristine-prednisone (Oncovin) reader. For consistency across experiments and to ensure a linear regimen (cyclophosphamide, doxorubicin, vincristine, and response between cell number and absorbance, the background- prednisone) is most frequently used (27). Mutations in TP53 corrected target absorbance value for untreated cells was kept at 0.9 in patients with non-Hodgkin’s lymphoma are associated with to 1.0 in all plates. Data were averaged and normalized against the a more aggressive clinical course (28) and relapse (29). nontreated controls to generate dose-response curves. Clin Cancer Res 2006;12(21) November 1, 2006 6548 www.aacrjournals.org Schedule-Dependent Synergy of DMAG and Doxorubicin Drug interaction analysis. The effects of drug combinations were Oncogene Research Products; CHK2 from Stressgen Biotechnologies evaluated using Calcusyn (BIOSOFT, Ferguson, MO), a software based (Victoria, BC); survivin from Novus Biologicals (Littleton, CO); CHK1, on the multiple drug-effect equation of Chou-Talalay (33). This method phospho-specific CHK1(S317), phospho-specific CHK2(T68), phos- defines the expected additive effect of agents used in combination and pho-specific p53(S15), HSP70, HSP90, cdc25C, phospho-specific then quantifies synergy or antagonism by determining how much the cdc25C(S216), AKT, and cleaved poly(ADP)ribose polymerase from combination effect differs from the expected additive effect. Values Cell Signaling Technology. Membranes were washed twice in TBS-T and of fraction affected (Fa) were calculated from cytotoxicity assays on incubated with species-specific horseradish peroxidase – labeled second- 96-well plates using the following formula: ary antibodies in TBS-T for 1 hour. Finally, the membranes were washed in TBS-T thrice and the proteins were visualized by enhanced ðbackground À corrected Abs490 treatedÞ chemiluminescence (Amersham, Piscataway, NJ). Fa ¼ 1À ðbackground À corrected Abs490 untreatedÞ Statistical analysis. Survival and apoptosis data are expressed as means F SD and means F SE, respectively. Statistical analysis was done These values represent the level of effect or survival loss achieved by with a two-tailed Student’s t test. each drug or drug combination. Such actual experimental data were entered into the Calcusyn interface and used to calculate combination index (CI) values, a quantitative measure of the degree of drug Results interactions for a corresponding Fa. Serial CI values over an entire range Functional status of p53 modulates the response to doxorubicin of drug-effect levels (Fa) were then calculated. These data were used to generate Fa-CI plots, from which synergy or antagonism can be and HSP90 inhibitors. We have previously shown that identified. Additivity, synergy, and antagonism are defined as CI = 1, doxorubicin-induced apoptosis is dependent on functional CI < 1, and CI > 1, respectively. p53 using p53-isogenic lymphoblastoid cell lines (26). TK6 Flow cytometry. Induction of apoptosis was evaluated through cells express WT p53. NH32 is a subclone of TK6 with a TP53- quantification of several variables by flow cytometry. Binding of targeted deletion and is p53-null (p53À/À). WTK1 was derived phosphatidyl-serine residues to Annexin V-FITC is indicative of from the same donor but expresses mutant p53 due to a early changes in cell membrane composition (Oncogene Research homozygous inactivating Met237Ile mutation in TP53 leading Products, Cambridge, MA). Positive staining with phycoerythrin- to protein accumulation and loss of transactivation (MT p53). conjugated anti – active caspase-3 antibody (measured by BD Bio- Doxorubicin exposure resulted in loss of cell viability in cells sciences, San Jose, CA) is indicative of caspase activation. Presence of with WT p53 TK6 cells with IC50 % 200 nmol/L, whereas cells with fragmented DNA is a later indicator of apoptosis. Annexin V and caspase-3 assays were done according to instructions from the IC50 > 2 Amol/L was observed for p53À/À NH32 or MT p53 manufacturers. For DNA analysis, cells were washed in ice-cold PBS WTK1 cells (Fig. 1A). A similar trend was observed in and fixed in 70% ethanol. Fixed cells were centrifuged at 1,200 rpm lymphoma cell lines (Fig. 1B), with IC50 % 200 nmol/L for for 5 minutes and stained by incubation in PBS containing 50 Ag/mL OCI-Ly3 and OCI-Ly10 (WT p53) and IC50 > 3 Amol/L for BJAB propidium iodide (Sigma) and 50 Ag/mL DNase-free RNase (Roche, and SUDHL-4 (MT p53; Fig. 1B). A consistent reduction in Indianapolis, IN). apoptotic markers, including staining with Annexin V-FITC, Histone H3 phosphorylation was evaluated using a phospho-specific and cleavage of poly(ADP)ribose polymerase, a surrogate antibody essentially as described (34). Briefly, ethanol-fixed cells were marker for caspase-3 activation, were observed in p53À/À and permeabilized in 0.25% Triton X-100/PBS on ice for 15 minutes, MT p53 cells (Supplementary Fig. S1). Doxorubicin exposure followed by blocking in 1% bovine serum albumin/PBS for 10 minutes also resulted in p53 protein phosphorylation and accumulation at room temperature. Cells were exposed to phospho-histone-H3 in WT p53 TK6 cells (Supplementary Fig. S1B). Analysis of (Ser10) antibody (Cell Signaling Technology, Danvers, MA) at 1:25 dilution or immunoglobulin G control in 1% bovine serum albumin/ p53 protein expression after exposure of SUDHL-4 cells to PBS for 30 minutes at room temperature. On washing, cells were doxorubicin showed a high basal level of p53 that was not exposed to FITC-conjugated goat anti-mouse antibody for 30 minutes increased by DNA damage (Supplementary Fig. S1C), consis- in the dark. Finally, they were washed and counterstained by tent with a p53-mutant phenotype. incubation in PBS containing propidium iodide and RNase. For all The presence of mutant p53 led to enhanced survival in assays, data were collected using FACScalibur from no less than 10,000 response to DMAG (Fig. 1C and D). Interestingly, lympho- cells with Cell Quest Pro software (BD Biosciences). blastoid cells were similarly sensitive to geldanamycin and Immunoblotting. Cell pellets were washed with ice-cold PBS and DMAG, but 10-fold higher doses of 17-AAG were required to lysed on ice in 50 mmol/L HEPES buffer containing 1% NP40, 150 achieve similar toxicities (Supplementary Fig. S2). These results mmol/L NaCl, 0.5 mmol/L EDTA (pH 8), 10% glycerol, 5 mmol/L sodium orthovanadate (pH 10), 10 mmol/L sodium fluoride, 2 mmol/L are in agreement with preclinical studies (21, 35) and indicate sodium pyrophosphate, and a protease inhibitor cocktail (Roche). that DMAG may have greater bioavailability than 17-AAG. After a 20-minute incubation, lysates were centrifuged at 14,000 rpm Based on this information, we did most of the experiments for 10 minutes, and the protein content of the supernatants was described below using DMAG. determined using a Bradford-based protein assay kit (Bio-Rad, Hercules, Schedule of administration determines the effect of doxorubicin CA). Aliquots of total cell protein (40-50 Ag/lane) were denatured by and DMAG on cell cycle progression. We evaluated the effect of boiling in loading buffer and loaded onto 8% or 10% gradient SDS- each drug and their combination on the cell cycle. Low indi- polyacrylamide gels (Invitrogen, Carlsbad, CA), depending on the sizes vidual doses of both drugs were selected from dose-response of target proteins. Proteins were separated by electrophoresis at 125 V curves (Fig. 1) aiming for >75% viability after 24-hour and then electrotransferred to nitrocellulose membranes at 30 V for exposure in all cell lines. The same low doses of 30 nmol/L 2 hours. Membranes were blocked at room temperature for 1 hour in TBS/0.05% Tween 20 (TBS-T) containing 5% nonfat dry milk doxorubicin and 100 nmol/L DMAG were chosen for these (monoclonal antibodies) or 5% bovine serum albumin (polyclonal experiments. Exposure to 100 nmol/L DMAG resulted in G1-S antibodies) and exposed to primary antibodies overnight at 4jC in arrest within 24 hours in lymphoblastoid and lymphoma blocking solution, with gentle rocking. The following commercial cell lines regardless of p53 status (Fig. 2A). These observa- primary antibodies were used: p53 (Ab-6) and actin (Ab-1) from tions are consistent with previously reported loss of pRb www.aacrjournals.org 6549 Clin Cancer Res 2006;12(21) November 1, 2006 Cancer Therapy: Preclinical Fig. 1. Status of p53 modulates the response to doxorubicin and DMAG. Cytotoxicity assay after 24 hours of exposure of lymphoblastoid (A) and lymphoma (B) cell lines to serial dilutions of doxorubicin (DOX). Cytotoxicity assay after 24-hour exposure of lymphoblastoid (C) and lymphoma (D) cell lines to serial dilutions of the HSP90 inhibitor DMAG. Survival was measured by standard MTS assay. Sextuplicate data were averaged and normalized against the nontreated controls to generate dose-response curves. Points, mean; bars, SD. Representative experiment. phosphorylation and G1 arrest in breast cancer cells after and that doxorubicin toxicity could be potentially enhanced treatment with 17-AAG (36). through bypass of G2-M checkpoint control promoted by A 24-hour exposure to 30 nmol/L doxorubicin led to 75% to DMAG. 80% viability in WT p53 cell lines and >90% viability in p53À/À DMAG enhances doxorubicin toxicity in a schedule-dependent and MT p53 cell lines. This modest effect on viability was manner. Next, we examined whether addition of DMAG to associated with major p53-dependent differences in cell cycle doxorubicin-primed cells would result in a lower IC50 for distribution. WT p53 TK6 and OCI-Ly3 cells accumulated doxorubicin in lymphoma cell lines. To this end, 100 nmol/L transiently with 2N and 4N DNA content (corresponding to DMAG was applied to cells that had been exposed for 24 hours G1-S and G2-M arrest, respectively) within 24 hours. After 48 to serial dilutions of doxorubicin. Survival was assessed by MTS hours, 15% (TK6) and 30% (OCI-Ly3) cells presented with sub- assay after a total of 48 hours. We found that sequential G1 DNA content, indicative of DNA fragmentation and addition of DMAG to doxorubicin-treated BJAB cells resulted in apoptosis. In contrast, p53À/À (NH32) and MT p53 (WTK1 a 5-fold reduction in IC50 for doxorubicin, from 200 nmol/L and BJAB) cells showed no G1-S arrest, rapid arrest in G2-M, (48-hour dose-response curve) to 40 nmol/L (Fig. 3A). and a negligible increase in the fraction of cells with sub-G1 To evaluate the role of schedule of exposure as a determinant DNA content after 48 hours (Fig. 2A and Supplementary of the overall response, we analyzed the interaction of Fig. S3). doxorubicin and DMAG on cell survival using a standard When 100 nmol/L DMAG was added to doxorubicin- criteria for synergy, the median effect/combination index exposed cells after a 24-hour delay, we observed loss of cells method (33). Survival was measured by sextuplicate using with G2-M DNA content and appearance of cells with standard MTS assay. CI values were calculated from experi- fragmented sub-G1 DNA content, regardless of p53 status mental data points and a Fa-CI plot was constructed by (Fig. 2A). In contrast, accumulation of cells in G1-S following simulating CI values over the entire range of Fa values from 5% exposure to DMAG was not altered by subsequent treatment to 95% using CalcuSyn software. Additivity, synergism, and with doxorubicin (Fig. 2B), indicating that sustained G1-S cell antagonism are defined as CI = 1, CI < 1, and CI > 1, cycle arrest induced by DMAG was protective from apoptosis. respectively. This software required a selection of a constant These data suggest that the outcome of DMAG and doxorubicin ratio for calculation of CI. Because we observed close to 80% combinations could be dependent on the schedule of exposure viability in all cell lines on exposure to either 30 nmol/L Clin Cancer Res 2006;12(21) November 1, 2006 6550 www.aacrjournals.org Schedule-Dependent Synergy of DMAG and Doxorubicin doxorubicin or 100 nmol/L DMAG, we selected a constant 1:3 although one set of experiments was carried out at a 1:1 molar ratio doxorubicin/DMAG for calculation of CI. Addition doxorubicin/DMAG molar ratio as well. We found that the of DMAG to doxorubicin-treated cells (doxorubicin!DMAG) sequential addition of DMAG to doxorubicin-treated cells was synergistic over an entire range of drug-effect levels, with was again highly synergistic (Table 1), with CI = 0.3 to 0.7 at CI = 0.4 at Fa = 0.5 indicative of a highly synergistic interaction Fa = 0.5. Schedule-dependent synergy was observed for both (Fig. 3B, open circles). Exposure to DMAG before doxorubicin WT p53 (OCI-Ly10 and OCI-Ly3) and MT p53 (BJAB and (DMAG!doxorubicin) not only showed lack of synergy but SUDHL-4) lymphoma cell lines. Similar schedule-dependent also resulted in an antagonistic effect (Fig. 3B, closed circles) synergy was observed when isogenic lymphoblastoid cell lines with CI > 2.0 at Fa = 0.5. The antagonistic effect of DMAG was were used (Table 1). Reversal in the order of drug exposure more evident at lower drug concentrations (Fa < 0.5), most resulted in nearly additive or antagonistic effects (CI = 0.8-5 at likely relevant for use in vivo (21). Simultaneous administration Fa = 0.5). The highest degree of antagonism was observed in of DMAG and doxorubicin resulted in additive or antagonistic MT p53 cell lines BJAB and SUDHL-4. effects as well (data not shown). HSP90 client protein expression in response to single drugs and To extend our observations, we exposed three other human drug combinations. To begin to examine the molecular basis lymphoma cell lines, SUDHL-4, OCI-Ly10, and OCI-Ly3, to for schedule-dependent synergy and antagonism between single drugs or combinations of doxorubicin and DMAG. The doxorubicin and DMAG, we analyzed the expression of heat drug molar ratio was maintained at 1:3 doxorubicin/DMAG, shock proteins and HSP90 clients after exposure of MT p53 Fig. 2. The effect of combination treatment with doxorubicin and DMAG on cell cycle progression depends on the schedule of administration. A, lymphoblastoid and lymphoma cell lines were exposed to 30 nmol/L doxorubicin for 24 hours before addition of 100 nmol/L DMAG and harvested 24 hours later for cell cycle analysis by staining with propidium iodide. B, BJAB cells were exposed to 100 nmol/L DMAG followed by 30 nmol/L doxorubicin with a 24-hour delay and harvested for cell cycle analysis. Percentages of cells with sub-G1 (fragmented DNA), G0-G1 (2N), and G2-M (4N) DNA content are indicated. www.aacrjournals.org 6551 Clin Cancer Res 2006;12(21) November 1, 2006 Cancer Therapy: Preclinical Fig. 3. Pretreatment with doxorubicin is required for synergy with DMAG. A, BJAB cells were exposed to serial dilutions of doxorubicin for 24 hours before addition of 100 nmol/L DMAG. Survival was measured 24 hours later using standard MTS assay. Sextuplicate data were averaged and normalized against untreated (doxorubicin) or DMAG-treated (doxorubicin!DMAG) controls. Points, mean; bars, SD. Note that the IC50 for doxorubicin shifts from 200 nmol/L (.) to 40 nmol/L (o) when 100 nmol/L DMAG is added sequentially after doxorubicin. B, BJAB cells were exposed to serial dilutions of a constant 1:3 doxorubicin/DMAG molar ratio with DMAG added with a 24 hours-delay (o, doxorubicin!DMAG) or with doxorubicin added with a 24-hour delay (., DMAG!doxorubicin). CI values were calculated from experimental survival data using Calcusyn Software and are shown as a function of Fa (fraction of cells killed by the combination). The Fa-CI plot was constructed by simulating CI values over the entire range of Fa values from 5% to 95% (lines) using CalcuSyn software.Three independent experiments for each schedule were combined to generate this plot. Synergism and antagonism are defined as CI < 1and CI > 1, respectively. C and D, analysis of protein expression comparing the two schedules of drug administration with 24- and 48-hour single treatments as above. BJAB cells to single agents and their combinations. Consistent cell cycle arrest and apoptosis (26). Therefore, we examined with earlier studies (35), exposure to DMAG effectively whether the combination with DMAG resulted in a schedule- inhibited HSP90, as evidenced by induction of HSP70, with a dependent increase in the apoptotic response of lymphoma minor effect on expression of HSP90 itself (Fig. 3C). The extent cells to doxorubicin. Results from the quantification of three of down-regulation of HSP90 client proteins was dependent on independent apoptotic markers are shown (Fig. 4). The earliest the length of exposure to DMAG. Specifically, loss of expression apoptotic marker studied was Annexin V (Fig. 4A). Exposure to of AKT, cdc25C, and CHK1 was observed as early as 24 hours 100 nmol/L DMAG alone resulted in 13% apoptosis at 24 on initial exposure, whereas loss of expression of survivin was hours and 27% at 48 hours. Exposure to 30 nmol/L only observed after 48 hours of exposure, and expression of doxorubicin alone resulted in 15% apoptosis at 24 hours and mutant p53 remained unchanged (Fig. 3C and D). 36% at 48 hours. Addition of DMAG to doxorubicin-primed Signal transduction pathways activated in response to cells with a 24-hour delay (doxorubicin ! DMAG) resulted in doxorubicin exposure involve ATM-dependent phosphoryla- 64% apoptosis, which is higher than expected for an additive tion of the checkpoint kinases CHK1 and CHK2 (37). In the response, and consistent with the schedule-dependent synergy absence of functional p53, these kinases are critical for observed in survival assays. In contrast, exposure to DMAG for maintaining G2-M arrest. Consistently, we found that CHK1 24 hours before doxorubicin (DMAG!doxorubicin) resulted and CHK2 were phosphorylated on Ser317 and Thr68, respec- in 32% apoptosis, which is less than expected for an additive tively, on exposure to doxorubicin (Fig. 3D). When DMAG was response. Similar results were obtained for caspase-3 activation added after doxorubicin, the levels of total and phosphorylated (Fig. 4B). Exposure to 100 nmol/L DMAG alone resulted in 6% CHK1 were substantially reduced, whereas only a small and 10% caspase-3 activation at 24 and 48 hours, respectively. decrease was observed in CHK2 activation (Fig. 3D). These Exposure to 30 nmol/L doxorubicin alone resulted in 15% and results indicate that synergy between DMAG and doxorubicin is 36% caspase-3 activation at 24 and 48 hours, respectively. associated with loss of G2-M checkpoint control through the Addition of DMAG to doxorubicin-primed cells (doxorubi- combined effect of HSP90 inhibition on the stability of CHK1 cin!DMAG) resulted in 55% caspase-3 activation, which is and possibly other HSP90 client proteins that participate on higher than expected for an additive response, whereas the cell cycle control after DNA damage. reverse schedule (DMAG!doxorubicin) resulted in 14% The apoptotic response to doxorubicin is enhanced by DMAG in caspase-3 activation, which is the expected value for an additive a schedule-dependent manner. Doxorubicin intercalates into interaction. The fraction of cells with sub-G1 DNA content is DNA to induce DNA damage and oxidative stress that result in shown (Fig. 4C). DNA fragmentation is the latest marker of Clin Cancer Res 2006;12(21) November 1, 2006 6552 www.aacrjournals.org Schedule-Dependent Synergy of DMAG and Doxorubicin Table 1. Schedule-dependent synergy of DMAG and doxorubicin Cell line p53 status Ratio doxorubicin/DMAG CI at IC50 Doxorubicin!DMAG DMAG!doxorubicin BJAB Mutant 1:3 0.4 F 0.1* 2.1 F 0.4* SUDHL-4 Mutant 1:3 0.4 5.3 1:1 0.5 8 OCI-Ly3 Wild-type 1:3 0.3 F 0.1* 0.8 F 0.6* OCI-Ly10 Wild-type 1:3 0.7 F 0.2* 3.8 TK6 Wild-type 1:3 0.3 1.3 NH32 Null 1:3 0.4 1.6 WTK1 Mutant 1:3 0.4 1.8 *Expressed as mean F SD from three independent experiments. apoptosis and lower overall values are anticipated. DNA has been used to identify mitotic cells and distinguish them fragmentation values are consistent with data obtained with from G2 cells in a flow cytometric assay (34). In this assay, the Annexin V and caspase-3. fraction of cells that incorporates propidium iodide with a G2 DMAG induces bypass of the G2-M checkpoint and premature DNA content and costains with anti – phospho-histone H3 mitosis. Altogether, our data indicate that addition of DMAG antibody is considered to be at the onset of mitosis. Expression to doxorubicin-primed cells enhances doxorubicin toxicity by of phospho-histone H3 was <1% in control or single-drug – forcing cells into premature mitosis in the presence of treated cells with G2 DNA content, but it increased to 18% unrepaired DNA damage. To test this hypothesis, we costained when DMAG was added to doxorubicin-treated cells (Fig. 5A). single-drug – treated and doxorubicin!DMAG – treated BJAB This data is consistent with abrogation of the doxorubicin- cells with an antibody specific for the phosphorylated form of induced G2-M checkpoint and premature mitotic entry. histone H3 and the DNA marker propidium iodide. Histone Inhibition of CHK1 sensitizes lymphoma cells to doxoru- H3 is phosphorylated exclusively during mitosis, and an bicin. Depletion of the HSP90 client CHK1 by 17-AAG has antibody that specifically recognizes its phosphorylated form previously been linked to increased sensitivity to DNA damage Fig. 4. The apoptotic response to doxorubicin is modulated by DMAG in a schedule-dependent manner. BJAB cells were exposed to 30 nmol/L doxorubicin or 100 nmol/L DMAG alone for 24 or 48 hours or to combinations in which the second drug was added with a 24-hour delay. Cells were harvested at indicated times and the frequency of three independent apoptotic markers was analyzed in parallel by flow cytometry. Dot plots depict Annexin V-FITC binding (A) and activation of caspase-3 (B). C, histograms depict the presence of cells with sub-G1 DNA content as detected by labeling with propidium iodide. The percentage of cells that is positive for each marker is indicated in the respective graphs. www.aacrjournals.org 6553 Clin Cancer Res 2006;12(21) November 1, 2006 Cancer Therapy: Preclinical (38 – 40). We hypothesized that depletion of active CHK1 on sensitivity of MT p53 cells to doxorubicin. Reversing the order exposure to DMAG might mediate sensitization of lymphoma of drug administration resulted in antagonistic effect in MT p53 cells to doxorubicin. To test this hypothesis, we exposed BJAB cells and only additive toxicity in WT p53 cells. These cells to increasing concentrations of the CHK1 inhibitor observations are particularly important for the design of phase SB-218078 (32), added 24 hours after doxorubicin. Exposure II clinical trials, which include patients with relapsed/refractory to SB-218078 alone in concentrations of up to 5 Amol/L disease, frequently bearing MT p53. produced negligible toxicity but significant G1-S arrest (Fig. 5B). It has been reported that HSP90 inhibitors sensitize cancer Addition of increasing concentrations of SB-218078 to doxo- cells to conventional therapies, including Taxol (41), gemcita- rubicin-treated cells caused a dose-dependent bypass of G2-M bine (38), cytarabine (40, 42), cisplatin (42), and radiation arrest and a concomitant dose-dependent increase in cells with (19, 43), and enhance apoptosis induced by molecularly fragmented DNA (Fig. 5B). Although expression and phos- targeted agents, such as imatinib (44, 45), SN38 (39), and phorylation of CHK2 were unaffected by SB-218078, dose- histone deacetylase inhibitors (46). It has been noted that cell dependent losses of CHK1 and phosphorylated CHK1 and of type and drug sequence influence the effect of HSP90 the CHK1 target cdc25C and its phosphorylated form were inhibition on the activity of cytotoxic drugs. HSP90 inhibitors observed (Fig. 5C). Cleavage of the caspase-3 substrate can be cytoprotective when administered together with or poly(ADP)ribose polymerase was also consistent with apopto- before chemotherapeutic agents, such as Taxol. It has been sis (Fig. 5C). These results indicate that inhibition of CHK1 speculated that induction of G1 arrest after exposure to HSP90 results in sensitization of lymphoma cells to doxorubicin and inhibitors leads to the cytoprotective effect when cells are support our hypothesis that CHK1 is a critical target of DMAG- pretreated with HSP90 inhibitors. Addition of 17-AAG to Taxol- mediated synergy. treated cells increased apoptosis, whereas exposure to 17-AAG before Taxol led to G1 arrest and protection from apoptosis in Discussion breast cancer cell lines (41). Similarly, we found that exposure to DMAG resulted in sustained G1 arrest that protected Our study addresses an important variable in clinical trial lymphoma cells from subsequent treatment with doxorubicin. design that is frequently empirically defined: how drug The cytoprotective effect of HSP90 inhibitors may also be schedule modulates the effect of DNA-damaging agents used mediated by HSP70 (47), presumably through its binding to in combination with molecularly targeted drugs. We found that apoptotic factors, leading to hindrance of one or more steps in synergy between doxorubicin and the HSP90 inhibitor DMAG the apoptotic cascade (48). Induction of HSP70 has been was schedule dependent and required pretreatment with observed in many cell types following inhibition of HSP90 and doxorubicin for 24 hours before the addition of DMAG. The is believed to be a consequence of transcriptional activation of combination of both drugs in this sequence enhanced the heat shock factor 1 (49). In our study, HSP70 was induced on Fig. 5. A, DMAG synergizes with doxorubicin by abrogation of doxorubicin- induced G2-M arrest. BJAB cells were pretreated with 30 nmol/L doxorubicin 24 hours before addition of 100 nmol/L DMAG. Cells were stained with phospho-histone H3/propidium iodide and analyzed by bivariate flow cytometry 24 hours later. The percentage of cells that is positive for phosphorylated histone-H3 is indicated in the respective graphs. B, inhibition of CHK1enzymatic activity sensitizes lymphoma cells to doxorubicin. BJAB cells were pretreated with 100 nmol/L doxorubicin, followed by increasing concentrations (50 nmol/L, 500 nmol/L, and 5 Amol/L) of the CHK1inhibitor SB-218078 24 hours later, and harvested for propidium iodide labeling and cell cycle analysis by flow cytometry. C, analysis of protein expression in BJAB cells pretreated with 100 nmol/L doxorubicin followed by increasing concentrations (50 nmol/L, 500 nmol/L, and 5 Amol/L) of the CHK1 inhibitor SB-218078 as above. Clin Cancer Res 2006;12(21) November 1, 2006 6554 www.aacrjournals.org Schedule-Dependent Synergy of DMAG and Doxorubicin exposure to DMAG regardless of sequence of administration, Abrogation of G2- and S-phase arrest by DMAG has and its expression was not associated with the overall response previously been associated with enhanced tumor cell radiosen- to drug combinations. We found in preliminary studies that sitivity, although no molecular mechanism was proposed to simultaneous administration of doxorubicin and HSP90 support this observation (43). We found that accumulation and inhibitors did not lead to synergistic toxicity (data not shown). phosphorylation of CHK1 observed on doxorubicin-induced Thus, we have no evidence that HSP70 exerts protection from G2-M arrest were substantially reduced when DMAG was apoptosis in lymphoma cells. administered 24 hours after doxorubicin. Furthermore, a It seems that the key to optimizing synergistic interactions specific CHK1 inhibitor added to doxorubicin-primed cells between DNA-damaging agents, such as doxorubicin, and reproduced DMAG-mediated synergy on induction of apopto- HSP90 inhibitors is to understand their effect on the signal sis. We propose that the main effect of HSP90 inhibition in the transduction pathways involved in cell cycle control. Many context of DNA damage-induced cell cycle arrest in lymphoma studies have addressed the role of p53 activation in response to cells is related to the abrogation of specific signal transduction radiation and chemotherapy (50 – 52). The signal transduction pathways essential for recovery from genotoxic stress. Apoptosis pathways activated by doxorubicin are well characterized (37) induced by doxorubicin is enhanced more effectively by HSP90 and evident in lymphoma cells used in this study. Doxorubicin inhibition in cells that lack functional p53 and must rely on G2- induced G1-S and G2-M arrest followed by apoptosis in WT p53 M checkpoint for DNA repair. cells, whereas MT p53 cells accumulated in G2-M. Because Molecularly targeted agents are likely to be used in HSP90 has numerous cellular clients (18), blockade of multiple combination with standard chemotherapeutic regimens for signal transduction pathways could contribute to sensitization patients with metastatic disease (14). Mutation and deletion of by HSP90 inhibitors. This complexity has hindered the p53 are frequently associated with resistance to therapy, understanding of molecular mechanisms of interaction. recurrence, and poor prognosis. It is timely to investigate how However, different client proteins exhibited different kinetics novel molecularly targeted agents interact with conventional of response to HSP90 inhibition. Although it has been therapies to sensitize tumors that lack functional p53. In our described as a HSP90 client (53), we did not observe loss of study, reduced sensitivity to doxorubicin- and DMAG-induced expression of MT p53 on exposure to DMAG. Survivin, a apoptosis was evident in MT p53 lymphoma cell lines. Similar protein involved in inhibition of mitochondrial apoptosis and dependency of HSP90 inhibitor – induced apoptosis on p53 regulation of mitosis, has also been described as a HSP90 status was reported in colon cancer cells exposed to geldana- client (54), and its degradation following 17-AAG has been mycin or 17-AAG (55). Here, we showed that synergy from the associated with sensitization of pediatric leukemia to imatinib combination doxorubicin and DMAG is determined by the (44). In our study, degradation of survivin required 48 hours schedule of exposure to each drug. Whereas schedule-dependent of exposure to DMAG and did not contribute to synergy synergy is independent of p53 status, antagonism in cells observed in the doxorubicin!DMAG combination. Further- pretreated with HSP90 inhibitors is more pronounced in cells more, loss of expression of survivin observed in the without functional p53. DMAG!doxorubicin combination did not lead to increased Altogether, our observations provide a rationale for further apoptosis, indicating that survivin may not be a critical target preclinical testing and stress the need to consider schedule of for DMAG-mediated sensitization in lymphoma. In contrast, exposure as a critical determinant of the overall response when degradation of other HSP90 clients, including AKT, CHK1, and designing clinical trials that combine DMAG with DNA- cdc25C, was evident within 24 hours of exposure to DMAG damaging agents for patients with relapsed/refractory disease. and correlated with increased toxicity drug combination. In this regard, CHK1, a key transducer of G2-M-phase arrest in Acknowledgments response to DNA damage (2), has previously been implicated in the sensitization to chemotherapeutic agents by HSP90 We thank Dr. Curtis C. Harris for his support and advice and Dr. CharlesVinson for inhibition (38 – 40). critical revision of the manuscript. References 1. Zhou BB, Elledge SJ. The DNA damage response: 8. Kawabe T. G2 checkpoint abrogators as anticancer neoplastic therapy: is it all wrapped up? Expert Opin putting checkpoints in perspective. Nature 2000; drugs. Mol Cancer Ther 2004;3:513 ^ 9. 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