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Clinical Cancer Research Vol. 12, 223-234, January 2006
© 2006 American Association for Cancer Research

Cancer Therapy: Preclinical

Evaluation of the Antitumor Efficacy, Pharmacokinetics, and Pharmacodynamics of the Histone Deacetylase Inhibitor Depsipeptide in Childhood Cancer Models In vivo

Claire Graham1, Chandra Tucker1, Jeremy Creech1, Edward Favours1, Catherine A. Billups2, Tiebin Liu2, Maryam Fouladi3, Burgess B. Freeman, III4, Clinton F. Stewart4 and Peter J. Houghton1

Authors' Affiliations: Departments of 1 Molecular Pharmacology, 2 Biostatistics, 3 Hematology-Oncology, and 4 Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee

Requests for reprints: Peter J. Houghton, Department of Molecular Pharmacology, St. Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105-2794. Phone: 901-495-3440; Fax: 901-495-4290; E-mail: peter.Houghton{at}stjude.org.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Histone acetyltransferases and histone deacetylases (HDAC) control the acetylation state of histones and other proteins regulating transcription and protein function. Several structurally diverse HDAC inhibitors have been developed as cancer therapeutic agents and in vitro have been shown to cause differentiation, cell cycle arrest, or apoptosis. Here, we have evaluated depsipeptide, a natural tetrapeptide HDAC inhibitor, against a panel of pediatric solid tumor models in vivo and evaluated pharmacokinetic and pharmacodynamic variables with tumor sensitivity.

Experimental Design: Depsipeptide was administered at the maximum tolerated dose (4.4 mg/kg administered every 7 days x 3 i.v. repeated q21d for a total of two cycles) to scid mice bearing 39 independently derived childhood tumors (9 brain tumors, 11 kidney cancers, 9 rhabdomyosarcomas, 3 neuroblastomas, and 7 osteosarcomas). Pharmacokinetic variables were determined, as were changes in histone and p53 acetylation, induction of p53 and p53 genotype, and alterations in Akt phosphorylation.

Results: Of 39 tumors evaluated, three showed objective tumor regressions [two brain tumors (primitive neuroectodermal tumor and atypical teratoid malignant rhabdoid tumor) and one Wilms' tumor]. Depsipeptide inhibited growth of many tumor lines but achieved stable disease (<25% increase in volume during treatment cycle 1) in only two tumor models (anaplastic astrocytoma, two rhabdomyosarcomas, and a Wilms' tumor). Pharmacokinetic analysis showed that the population estimated AUC0-24 was 1,123 ng h/mL, similar to the exposure following 13 mg/m2 in ongoing phase I trials. Pharmacodynamic changes in histone acetylation (H2A, H2B, H3, and H4) in three depsipeptide-sensitive and three intrinsically resistant tumors followed a similar pattern; maximal increases in histone acetylation occurred at 8 hours and were elevated for up to 96 hours. In two sensitive tumor lines, IRS56 and BT27 (both wild-type p53) p53 increased in treated tumors being maximal at 8 hours and associated with induction of p21cip1, whereas p53 was stable in tumors with mutant p53. Sensitivity to depsipeptide did not correlate with p53 genotype, p53 acetylation, cleaved poly(ADP-ribose) polymerase, or phosphorylation of Akt (Ser473).

Conclusions: Our results show that depsipeptide inhibits its target in vivo causing increased histone acetylation; however, this does not correlate with drug sensitivity. The relatively low objective response rate [3 of 39 (8%) tumor lines showing greater than or equal to partial response and 4 (10%) stable disease] administered at dose levels that give clinically relevant drug exposures suggests that as a single agent depsipeptide may have limited clinical utility against pediatric solid tumors in a first-line setting.

Chromatin consists of nucleosomes comprising 146 bp of DNA wound around a histone octamer core. Core histones are arranged as a (H3-H4)2 tetramer and two H2A-H2B dimers positioned on both sides of the tetramer. Core histones have a similar structure consisting of a basic NH2-terminal domain, a globular histone fold domain mediating histone-histone and histone-DNA interactions, and a COOH-terminal tail (1). Specific lysines in the NH2-terminal domains of core histones are susceptible to reversible acetylation. Core histones are also susceptible to other modifications (i.e., methylation, phosphorylation, and ubiquitination; refs. 2, 3).

Histone acetyltransferases catalyze acetylation of the NH2-terminal lysine residues in histones, which neutralizes the positive charge leading to a more open conformation of chromatin. The more open conformation of chromatin may allow greater access to DNA-binding transcription factors, thereby allowing transcription. Histone deacetylases (HDAC) remove the acetyl groups, thus restoring the positive charge and leading to chromatin condensation and repression of transcription (4, 5). There is growing evidence that histone acetyltransferases play a role in regulating tumorigenesis and that alterations in histone acetylation and methylation occur frequently in human cancers (6–8).

Several structurally diverse HDAC inhibitors (HDACI) have been developed, which induce either differentiation, cell cycle arrest in G1 or G2-M, or apoptosis of various tumor cell types with little or no toxicity against normal cells (9). However, global hyperacetylation evoked by HDACIs does not induce a general increase of gene transcription. Depsipeptide (FK228/FR901228) is a natural tetrapeptide that was isolated from Chromobacterium violaceum no. 968 and was found to potently inhibit class 1 HDACs (10). Depsipeptide strongly inhibits HDAC1 and HDAC2 but only weakly affects HDAC4 and HDAC6 (9) and potently inhibited the proliferation of tumor cells in vitro by arresting cell cycle transition at G1 and G2-M phases (11, 12) and induced the expression of the CDKN1A gene, which encodes the cyclin-dependent kinase inhibitor p21cip1 (13). This agent caused apoptosis in several human tumor cells as defined by cell shrinkage and internucleosomal breakdown of DNA (11).

In vivo depsipeptide suppressed the growth of transplanted tumors (14) and has been shown to inhibit hypoxia-stimulated angiogenesis. Depsipeptide and other HDACIs have been evaluated previously against pediatric cancer cell lines (15–17) and in a limited spectrum of xenograft models (17). Depsipeptide may be a potentially useful drug in the treatment of pediatric solid malignancies; therefore, we have investigated the single-agent activity against 39 pediatric tumor models growing as xenografts in scid mice. Another aim of this study was to determine whether pharmacodynamic changes in tumor tissue were predictive of tumor response to treatment.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Growth inhibition studies. CB17/Icr female scid–/– mice (Charles Rivers, Wilmington, MA) were implanted s.c. with a single tumor fragment as described previously (18, 19). Mice bearing s.c. tumors each received depsipeptide when tumors were ~0.20 to 1 cm in diameter. The procedures have been reported previously (20). Tumor-bearing mice were randomized into groups of 5 to 10 before therapy. All mice were maintained under barrier conditions. All experiments were conducted using protocols and conditions approved by the St. Jude Children's Research Hospital Institutional Animal Care and Use Committee.

Tumor models. Depsipeptide was evaluated against 39 independently derived childhood tumors (9 brain tumors, 11 kidney cancers, 9 rhabdomyosarcomas, 3 neuroblastomas, and 7 osteosarcomas) growing in scid mice as s.c. xenografts as summarized previously (21). Kidney tumors and brain tumors not reported previously will be described elsewhere.5 Mice with xenografts (~0.5 cm3) were treated i.v. with depsipeptide at the maximum tolerated dose level (4.4 mg/kg administered every 7 days x 3 i.v. with a second cycle of treatment starting on day 21). Prior experiments had determined that this schedule and dose was optimal in several tumor models. Tumor volumes were measured for up to 12 weeks.

Tumor response and tumor failure time. For individual tumors, partial response was defined as a volume regression of >50% but with measurable tumor (≥0.10 cm3) at all times. A complete response (CR) was defined as a disappearance of measurable tumor mass (<0.10 cm3) at some point within the study period after initiation of therapy. Maintained CR was a CR without tumor regrowth at the end of the 12-week study period. Tumor failure time was defined as the time (in weeks) required by individual tumors to quadruple their volume from the initiation of therapy. Tumor failure times were said to be censored if a mouse died before week 12 and before a tumor grew to four times its initial volume.

Statistical methods. For comparisons of time to tumor failure, survival distributions of each treatment group were compared with the survival distribution of the control group using the exact log-rank test. Given the exploratory nature of this study, the Ps presented were not adjusted for multiple comparisons. Ps < 0.05 were considered to be statistically significant.

Drugs and formulation. Depsipeptide was generously provided by Fujisawa Co. (Osaka, Japan) and was formulated in ethanol (40%)-polyethylene glycol 400 (5%) in saline and administered i.v. (0.05 mL/10 g body weight) at a dose of 4.4 mg/kg as a short injection (duration of administration was <1 minute) into lateral tail vein. Mice received three administrations of each agent at 7-day intervals (abbreviated q7d x 3) with a second cycle of treatment starting on day 21.

Pharmacokinetic studies. Depsipeptide pharmacokinetics were evaluated in non-tumor-bearing scid mice after a single i.v. bolus of 4.4 mg/kg. Three mice contributed one plasma sample via terminal cardiac puncture at each of the following time points after administration: 10, 30, 60, 120, and 240 minutes. Plasma concentrations of depsipeptide were assessed using a previously described validated solid-phase extraction, liquid chromatography, tandem mass spectrometry assay (22). Briefly, this assay had a lower limit of quantitation of 0.2 ng/mL and showed good within-day and between-day precision (CV% values were ≤3.5% and ≤5.5%, respectively) and accuracy (range, 99.7-112.5%) across the calibration range.

Murine plasma depsipeptide concentration-time data were evaluated using compartmental techniques and maximum likelihood estimation as implemented in ADAPT II (23). Estimated model variables included the volume of the central compartment (Vc), elimination rate constant (ke), and intercompartment rate constants (kcp and kpc). Systemic clearance and terminal half-life (T1/2ß) were calculated using the model variables. To quantitate murine depsipeptide exposure at this dosage, the area under the depsipeptide concentration-time curve from 0 to 24 hours (AUC0-24) was calculated using the variable estimates and the log-linear trapezoidal method.

Pharmacodynamic studies. Three sensitive tumors (CR, BT27, and two with stable disease, BT35 and IRS56) and three resistant tumors (SKNEP, GBM2, and WT10) were grown as xenografts in scid mice. Tumors (~0.5 cm3) were removed and immediately placed in liquid nitrogen 0, 4, 8, 24, 48, 96, and 168 hours after i.v. administration of 4.4 mg/kg depsipeptide. Tumors were stored at –80°C.

Western blot analysis. Tumors were ground to powder under liquid nitrogen, and ~100 mg were placed in 1 mL lysis buffer (0.02 mol/L Tris, 0.2 mmol/L Triton X-100, 0.02% 2-mercaptoethanol, One Complete Mini tablet/10 mL) and heated at 100°C for 5 minutes. Protein concentration was determined using bicinchoninic acid protein assay (Pierce, Rockford, IL) and diluted to 30 µg/20 µL in lysis buffer containing SDS sample buffer and heated at 100°C for 5 minutes. Total cellular protein extracts (30 µg) were separated using the NuPAGE Bis-Tris electrophoresis system (Invitrogen, Carlsbad, CA) and transferred to polyvinylidene difluoride membrane in NuPAGE transfer buffer containing 20% (v/v) methanol. Membranes were blocked with 5% nonfat dry milk in 1x TBS containing 0.05% Tween 20 and incubated with antibody specific for acetylated forms of histones H2A, H2B, and H4 (Upstate Biotechnology, Lake Placid, NY), p53, acetyl p53 (Lys382), p21WAF1/cip1, Akt, phosphorylated Akt (Ser473), cleaved poly(ADP-ribose) polymerase (PARP; Asp214), HDAC1 (Cell Signaling Technology, Beverly, MA), HDAC2 (Upstate Biotechnology), and HDAC3, HDAC4, HDAC5, HDAC6, and HDAC7 (Cell Signaling Technology). ß-Tubulin (Sigma, Atlanta, GA) was used to ensure equal loading and transfer of proteins. Protein bands were detected by incubating with horseradish peroxidase–conjugated antibodies (Amersham Biosciences, Little Chalfont, United Kingdom). Acetylated and nonacetylated histone bands were visualized with Enhanced Chemiluminescence Plus (Amersham Biosciences) on the PhosphorImager Storm 860 (GE Healthcare, Piscataway, NJ); all other proteins were visualized with SuperSignal West Pico Chemiluminescent Substrate (Pierce).

Sequencing of p53 mRNA. RNA was isolated from control tumor powder (70-180 mg) using Trizol reagent (Invitrogen) according to the manufacturer's instructions. Reverse transcription-PCR (RT-PCR) was carried out using the Qiagen One-Step RT-PCR kit (Qiagen, Valencia, CA). A mixture containing 10 µL of 5x Qiagen One-Step RT-PCR buffer, 2 µL deoxynucleotide triphosphate mix, 0.6 µmol/L forward PCR primer, 0.6 µmol/L backward PCR primer, 2 µL Qiagen One-Step RT-PCR Enzyme Mix, 1.5 to 1.8 µg RNA, and volume made up to 50 µL with RNase-free water. Samples were placed in the thermal cycler (Eppendorf, Hamburg, Germany) under the following conditions: 30 minutes at 50°C, 15 minutes at 95°C, 45 cycles of 1 minute at 94°C, 1 minute at 65°C, and 1.5 minutes at 72°C, and finally 10 minutes at 72°C.

RT-PCR products were electrophoresed on a 0.8% agarose gels, bands at ~1.2 kDa were excised, and DNA was extracted using QIAquick Gel Extraction kit according to the manufacturer's instructions. Six sequencing reactions were set up per tumor containing 3.2 pmol primer (Table 1) and 50 ng DNA made up to a final volume of 12 µL with DNase, RNase-free water. DNA sequencing was done by the Center for Biotechnology at St. Jude Children's Research Hospital using automated sequencing.


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  		Table 1. Primers for RT-PCR and sequencing

 

   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Growth inhibitory studies. A diverse range of pediatric cancers was grown as xenografts in scid mice that were treated with depsipeptide 4.4 mg/kg administered q7d x 3 i.v. (repeated q21d). Of 39 tumors treated, 6 responded to therapy; 3 tumor lines showed partial or complete regressions in virtually all mice in multiple experiments. Results for different tumor types are summarized in Table 2. Data presented show drug-induced growth inhibition, treated versus control tumor volume ratios at the end of the first cycle of treatment, and frequency of regressions for each tumor line. Responsive tumors included two brain tumors (BT27, a primitive neuroectodermal tumor, and BT40, an atypical teratoid malignant rhabdoid tumor as well as WT8, a Wilms' tumor nephroblastoma of favorable histology; Fig. 1A). Depsipeptide induced stable disease (defined as <25% increase in tumor volume at the end of the first drug cycle at day 21) in four additional tumor lines [BT35 (anaplastic astrocytoma), IRS56, Rh66 (embryonal and alveolar rhabdomyosarcomas, respectively), and WT6 (Wilms' tumor)]. For each line, there were sporadic partial responses and CRs; however, within any treatment group, there were tumors that did not reach the definition of partial response. Typical responses for xenografts where depsipeptide induced stable disease are shown in Fig. 1B. No significant difference in growth of tumor was observed between control and depsipeptide-treated mice bearing SKNEP (diffuse anaplastic Wilms' tumor), GBM2 (glioblastoma), and WT10 xenografts (favorable histology Wilms; treated versus control = 0.63, 1.01, and 0.93, respectively; Fig. 1C); hence, these tumors were considered intrinsically resistant. Three sensitive tumors BT27 (CR) and BT35 and IRS56 (stable disease) and three resistant tumors SKNEP, GBM2, and WT10 were chosen for further pharmacodynamic and biochemical investigation.


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  		Table 2. Summary of responses of xenografts to depsipeptide

 



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  		Fig. 1. A, responses of depsipeptide-sensitive xenografts. Top, BT27 (primitive neuroectodermal tumor); middle, BT40 (atypical teratoid malignant rhabdoid tumor); bottom, WT8 (Wilms' tumor) xenografts. Tumor volume was measured for up to 12 weeks. a, c, and e, control tumors; b, d, and f, depsipeptide (4.4 mg/kg) was administered q7d x 3 i.v. (repeated q21d x 2). Each curve represents the growth of an individual tumor. B, responses of xenografts showing moderate sensitivity to depsipeptide. Top, BT35 (anaplastic astrocytoma); middle, IRS56 (embryonal rhabdomyosarcoma); bottom, Rh66 (alveolar rhabdomyosarcoma) xenografts. Tumor volume was measured for up to 12 weeks. a, c, and e, control tumors; b, d, and f, depsipeptide (4.4 mg/kg) administered q7d x 3 i.v. (repeated q21d x 2). Each curve represents the growth of an individual tumor. C, responses of xenografts intrinsically resistant to depsipeptide. Top, GBM2 (glioblastoma); middle, WT10 (favorable histology Wilms' tumor); bottom, SKNEP (diffuse anaplastic Wilms' tumor) xenografts. Tumor volume was measured for up to 12 weeks. a, c, and e, control tumors; b, d, and f, depsipeptide (4.4 mg/kg) administered q7d x 3 i.v. (repeated q21d x 2). Each curve represents the growth of an individual tumor.

 
Pharmacokinetic studies. To determine whether the dose of depsipeptide used in mice produced similar systemic exposure to that determined in clinical trials, detailed pharmacokinetic studies were undertaken. The murine plasma depsipeptide concentration-time data were best described using a two-compartment model. Figure 2 depicts the depsipeptide concentration-time profile after a dose of 4.4 mg/m2 in non-tumor-bearing scid mice. The murine pharmacokinetic results are compared with those from adults (23, 24) and pediatric patients6 in Table 3. These results indicate that similar systemic exposure to that reported in adults was achieved in mice at the dose of depsipeptide used.



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  		Fig. 2. Depsipeptide pharmacokinetics in nontumored mice. Depsipeptide concentration-time profile after administration of 4.4 mg/kg i.v. in non-tumor-bearing scid mice. Solid circles, actual data points; solid line, best-fit line from the pharmacokinetic analysis.

 

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  		Table 3. Comparison of murine depsipeptide pharmacokinetics with results of adult and pediatric pharmacokinetic studies

 
HDAC expression. We next examined the levels of class 1 HDACs (HDAC1, HDAC2, and HDAC3) and class 2 HDACs (HDAC4, HDAC5, HDAC6, and HDAC7) relative to ß-tubulin in sensitive and resistant tumors, respectively (Fig. 3A and B). Histone H2 deacetylase was the predominant class 1 isoform in all tumors irrespective of depsipeptide sensitivity. BT27 tumors, which respond completely to depsipeptide, expressed predominantly histone H2 deacetylase and expressed low levels of the class 2 HDACs. BT35 tumors had similar levels of expression of HDACs to those seen with BT27 tumors, whereas IRS56 tumors had similar levels of HDACs to resistant tumors (SKNEP, GBM2, and WT10; Fig. 3B), except the expression levels of HDAC 6 were much higher in IRS56 tumors compared with the other tumors. The pattern of expression of HDACs does not correlate with which tumor types respond to depsipeptide therapy.



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  		Fig. 3. HDAC levels and pharmacodynamic changes in depsipeptide-responsive and depsipeptide-resistant xenografts. Tumors were grown as xenografts in scid mice and removed once they reached 0.5 cm3 and protein was isolated. Protein was subjected to Western blot assay using antibodies against HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, and ß-tubulin. Membranes were visualized on the PhosphorImager Storm 800 and a ratio to ß-tubulin was determined. Columns, mean of three separate experiments; bars, SD. Sensitive (BT27, BT35, and IRS56; A) and resistant (SKNEP, GBM2, and WT10; B) tumors. C, depsipeptide caused an increase in the acetylation state of core histones. Tumors were removed 0, 4, 8, 24, 48, 96, and 168 hours after depsipeptide treatment and protein was isolated. Protein was subjected to Western blot assay using antibodies against acetyl histones H2A, H2B, H3, and H4 and ß-tubulin. Membranes were visualized on the PhosphorImager Storm 800, normalized to ß-tubulin, and compared with control to determine fold increase. Points, mean of three separate experiments; bars, SD. BT27 ({blacklozenge}), BT35 ({blacksquare}), IRS56 ({blacktriangleup}), SKNEP (x), GBM2 ({square}), and WT10 ({circ}) growing as xenografts. Tumors were removed 0, 4, 8, 24, 48, 96, and 168 hours after depsipeptide treatment and protein was isolated. Protein was subjected to Western blot assay using antibodies against acetyl histone H2A, H2B, H3, and H4 and ß-tubulin. Membranes were visualized on the PhosphorImager Storm 800, normalized to ß-tubulin, and compared with control to determine fold increase. Points, mean of three separate experiments; bars, SD. D, tumors were removed 0, 4, 8, 24, 48, 96, and 168 hours after depsipeptide treatment and protein was isolated. Protein was subjected to Western blot assay using antibodies against acetyl histone H4 (Ac-H4) and ß-tubulin. Membranes were visualized on the PhosphorImager Storm 800. Each lane was loaded with 30 µg protein and membrane was probed with ß-tubulin to ensure equal loading. Two additional studies yielded equivalent results.

 
Pharmacodynamic studies: acetylation of histones. Mice bearing BT27, BT35, IRS56, SKNEP, GBM2, and WT10 xenografts (~0.5 cm3) were treated i.v. with depsipeptide (4.4 mg/kg). For each xenograft line, tumors (three per time point) were excised 0, 4, 8, 24, 48, 96, and 168 hours after treatment and protein was extracted for Western blot analysis of the acetylation state of core histones (H2A, H2B, H3, and H4). Levels and changes in H4 acetylation for depsipeptide-sensitive (BT27, BT35, and IRS56) and depsipeptide-resistant (GBM2, SKNEP, and WT10) tumors are presented in Fig. 3A-D. Depsipeptide caused the greatest degree of acetylation of all the core histones in BT35 tumors. In this "sensitive" tumor, acetylation of H2A, H2B, H3, and H4 remained significantly elevated relative to control tumors for at least 96 hours after administration of drug. In contrast, in all the other tumors, there was a less pronounced effect on core histone acetylation, irrespective of tumor sensitivity, and a very similar degree of acetylation in all the core histones. Histone acetylation peaked in all tumors at 8 hours after depsipeptide treatment. All histones remained acetylated for up to 96 and 168 hours after treatment in the IRS56 and BT35 tumors, respectively, whereas this was maintained for only 24 tumors in GBM2 and WT10 and 48 hours in SKNEP tumors. The kinetics of H4 acetylation following depsipeptide administration for different tumor models are presented in Fig. 3D. Thus, the increased level and duration of histone acetylation does not seem to predict whether a tumor is sensitive to depsipeptide. However, increased acetylation of histones in vivo indicates that depsipeptide reaches it target in sufficient concentration to inhibit HDACs.

p53 status and protein levels of tumors. In vitro, depsipeptide has been reported to induce p53-dependent expression of the cyclin-dependent kinase inhibitor p21cip1. We therefore determined whether depsipeptide induced p53 protein levels in sensitive and resistant tumors by Western blot analysis (Fig. 4A and B). p53 was barely detected in BT27 and IRS56 tumors that are depsipeptide sensitive but was induced by treatment (Fig. 4A). In contrast, p53 levels were readily detected in sensitive (BT35) and resistant (SKNEP, GBM2 and WT10; Fig. 4B) tumors. The presence of p53 in the control tumors would suggest these tumors have mutant p53. The p53 genotype was confirmed by RT-PCR and DNA sequencing (Table 4). WT10 tumors had a lower level of p53 expression than the other mutant p53 tumors, which may be a consequence of heterozygosity at position 290 (Arg or His/wild-type or mutant). Acetylation of p53 at Lys373 and Lys382 in response to DNA damage has been reported (25). We determined whether depsipeptide induced acetylation of p53 (Lys382) in each of the tumors (Fig. 4). Depsipeptide induced transient acetylation of p53 in "sensitive" tumors, consistent with the drug-induced transient increase in detectable p53 protein. In contrast, acetylated p53 was detected in SKNEP and GBM2 for at least 96 hours, whereas the effect of depsipeptide was transient in WT10 xenografts.



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  		Fig. 4. Stabilization and acetylation of p53 in treated tumors and p21cip1 induction. Depsipeptide (4.4 mg/kg) was administered i.v. to scid mice. Tumors were removed 0, 4, 8, 24, 48, 96, and 168 hours after depsipeptide treatment and protein was isolated. Protein was subjected to Western blot assay using antibodies against p53 and acetyl p53 (Ac-p53; Lys382), p21cip1, and ß-tubulin. Each lane was loaded with 30 µg protein and membrane was probed with ß-tubulin to ensure equal loading. Two additional studies yielded equivalent results. Sensitive (BT27, BT35, and IRS56; A) resistant (SKNEP, GBM2, and IRS56; B) tumors growing as xenografts. C, induction of p21cip1 in depsipeptide-treated xenografts.

 

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  		Table 4. p53 status and mutations of tumors differing in sensitivity to depsipeptide

 
Induction of p21. Depsipeptide caused an induction in p21cip1 in BT27 and IRS56 tumors with wild-type p53 (Fig. 4C), whereas none was detected in the other tumors with mutant p53 (data not shown). In the BT27 tumors, p21cip1 reached a peak 8 hours after depsipeptide treatment and levels remained above control levels for the remainder of the time course. p21cip1 also reached a peak 8 hours after depsipeptide in the IRS56 tumors, but levels were not detectable by 24 hours.

Cleavage of PARP. Depsipeptide increased the cleavage of PARP to its 87-kDa fragment in sensitive tumors BT27, IRS56 (Fig. 5A) but also in resistant SKNEP and GBM2 xenografts (Fig. 5B). The kinetics of enhanced detection of cleaved PARP was similar between BT27 and GBM2 and IRS56 and SKNEP. In the former pair, there was a transient increase in cleaved PARP being maximal at 24 hours, whereas in the latter pair cleaved PARP was elevated for up to 96 hours. No cleaved PARP was detected in BT35 and WT10 tumors (data not shown).



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  		Fig. 5. Cleavage of PARP or phosphorylation of Akt does not correlate with tumor response to depsipeptide. Depsipeptide (4.4 mg/kg) was administered i.v. to scid mice. Tumors were removed 0, 4, 8, 24, 48, 96, and 168 hours after treatment and protein was isolated. Protein was subjected to Western blot assay using antibodies against cleaved PARP (cPARP), Akt and phosphorylated Akt (Ser473), and ß-tubulin. Each lane was loaded with 30 µg protein and membrane was probed with ß-tubulin to ensure equal loading. Two additional studies yielded equivalent results. Depsipeptide causes an increase in the cleaved fragment of PARP (cPARP; 87 kDa) in both sensitive and resistant tumors. A, PARP cleavage in sensitive and resistant tumors. B, changes in pAkt in sensitive tumors. C, changes in pAkt in resistant tumors.

 
Phosphorylation of Akt. Recently, Kodani et al. (26) concluded that depsipeptide suppresses the phosphatidylinositol 3-kinase/Akt signaling pathway in a cell-specific manner, and this effect is a determinant of sensitivity to this agent. The levels of Akt did not differ between sensitive and resistant tumors and over the time course examined after depsipeptide treatment remained stable in all the tumors studied (Fig. 5C). Phosphorylated Akt (pAkt; Ser473) was detected in untreated tumors (BT35, IRS56, SKNEP, and GBM2) but not in BT27 or WT10. In the most sensitive tumor, BT27, the phosphorylation of Akt (Ser473) was low or undetected for 24 hours following depsipeptide treatment but subsequently increased for up to 168 hours. In other tumors, the changes in pAkt were relatively subtle, except in the WT10 Wilms' tumors where there was a consistent increase in pAkt at 48 hours only.


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
HDACIs have shown significant activity against many cell lines in vitro and have been shown to inhibit growth of xenograft tumors growing s.c. or orthotopically in immune-deficient rodents (14–18). Depsipeptide, a natural tetrapeptide that was isolated from C. violaceum, has recently entered phase I testing in pediatric patients. However, there is little preclinical data to date to support the development of HDACIs for childhood malignancies. Jaboin et al. (17) determined the sensitivity of pediatric cancer cell lines exposed to MS-275, a synthetic benzamide derivative, in vitro and in several orthotopic models in scid/beige mice. Cell line sensitivity varied over several orders of magnitude (IC50, 70-1,300 nmol/L), and MS-275 induced apoptosis in cell lines irrespective of p53 status. In contrast, NIH3T3 immortalized fibroblasts were resistant to this agent (IC50 > 10,000 nmol/L). In vivo, however, MS-275 retarded tumor growth, leading to stable disease in three tumor models [US (undifferentiated sarcoma), TC71 (Ewing sarcoma), and KCNR (neuroblastoma)]. Similar to MS-275, depsipeptide inhibits growth and induces apoptosis of human cancer cell lines in vitro and inhibits growth in vivo, whereas it seems relatively nontoxic to fibroblasts in vitro. To gain insight into the antitumor activity of depsipeptide, we tested it against a panel of 39 pediatric tumors representing brain tumors, kidney cancers, rhabdomyosarcomas, neuroblastomas, and osteosarcomas growing as xenografts in scid mice. The maximum tolerated dose [22 of 465 (4.7%) drug-related deaths] on this schedule was 4.4 mg/kg administered weekly for 3 consecutive weeks, with a second cycle beginning day 21. Seven tumors (three brain tumors, two kidney cancers, and two rhabdomyosarcomas) responded to depsipeptide treatment showing volume regressions [3 of 39 (8%)] or stable disease [4 of 39 (10%)]. Notably, although depsipeptide retarded growth ~50% (3 of 4 neuroblastomas, 2 of 6 osteosarcomas), growth of these tumors exceeded 25% during the first cycle of treatment and hence may be considered progressive disease. Thus, as a single agent, depsipeptide has only moderate activity against this panel of tumor models. In contrast, agents, such as topotecan, irinotecan, and standard agents, used in treatment of many pediatric solid tumors (vincristine, cyclophosphamide, actinomycin D, and doxorubicin) show higher response rates in the neuroblastoma, rhabdomyosarcoma models (27, 28).

Clearly, such xenograft models may either overpredict or underpredict the activity of a drug. In part, this results from differences in host tolerance (29). For example, the acylfulvene MGI-114 shows dramatic antitumor activity in a wide panel of xenografts but only at dose levels associated with systemic exposures of drug that significantly exceed those achieved clinically (18). To determine whether systemic exposures of depsipeptide in mice were relevant to clinical studies, depsipeptide pharmacokinetics were evaluated in non-tumor-bearing scid mice. Many similarities between murine and human pharmacokinetics were noted; specifically, clearance and volume of central compartment values were concordant between species. Notably, in a phase I study of depsipeptide in children with refractory solid tumors, the mean AUC0-24 observed at the maximum tolerated dosage (i.e., 17 mg/m2) was greater than that observed in mice given dosages found effective against pediatric tumor xenografts (2,444 versus 1,123 ng/mL h, respectively). Thus, the dose level used in this preclinical study seems appropriate and is associated with clinically relevant systemic exposure, although slightly lower than that in patients.

Depsipeptide strongly inhibits HDAC1 and HDAC2 but only weakly affects HDAC4 and HDAC6 (9); thus, relative levels of histones were examined in depsipeptide-sensitive and depsipeptide-resistant tumors. Of the tumors examined, the most sensitive tumor (BT27) expressed lower levels of HDACs, except HDAC2, than the other tumors studied. Therefore, one may speculate that in BT27 tumors HDAC2 may be most prevalent in the removal of acetyl groups, so inhibition with depsipeptide, which in vitro strongly inhibits HDAC2, may have more of an effect on the deacetylase activity of this tumor type. However, determination of HDAC activity in the presence of inhibitor would be required to support such conjecture. In vitro, depsipeptide (5 nmol/L) increased histone acetylation after 24 hours in DLD-1 cells (30) and increased acetylation was due to inhibition of histone deacetylation, which was confirmed by determining the effects on HDAC activity (10, 11). Therefore, the level of histones and histone acetylation state was investigated in tumors that were sensitive and insensitive to depsipeptide treatment. Depsipeptide caused an increase in the acetylation state of the core histones in both sensitive and resistant tumors. The BT35 tumor had the greatest increase in acetylation state of all the core histones, whereas the most sensitive line, BT27, had less pronounced changes in acetylation of histones. Thus, the level and duration of acetylation does not seem to predict whether a tumor is sensitive to depsipeptide. These results, however, do show that depsipeptide achieve sufficient concentration in tumors to inhibit HDACs.

In vitro inhibition of HDACs has been shown to induce p21cip1 in a p53-dependent manner, resulting in cell cycle arrest and apoptosis, although for some cell lines induction of apoptosis seems to be p53 independent (9). p53 is a short-lived protein that is maintained at low, often undetectable, levels in normal cells (31) and becomes stabilized following DNA damage. Previous studies using mouse embryonic fibroblasts reported that p53 wild-type mouse embryonic fibroblasts were sensitive to the HDACIs trichostatin A (50ng/mL) and sodium dibutyrate (0.25 mmol/L), whereas p53–/– mouse embryonic fibroblasts were completely resistant to trichostatin A (100 ng/mL) and sodium dibutyrate (0.5 mmol/L) had only a slight effect (32). Consequently, we examined induction of p53 following depsipeptide, and for each of the three depsipeptide-sensitive and depsipeptide-resistant tumors, the p53 genotype was determined (Table 4).

Two sensitive tumors (BT27 and IRS56) had wild-type p53, and p53 was increased after depsipeptide treatment. Stabilization of p53 after depsipeptide treatment was not due to phosphorylation at Ser15 (data not shown) but seemed to be due to acetylation. Acetylation of p53 by CBP/p300 (histone acetyltransferases) has been shown to potentiate its transcriptional activity in vivo (33, 34) and in vitro could dramatically increase the DNA-binding activity of p53 (34). However, in our study, p53 genotype did not predict responsiveness to depsipeptide; several other tumors with wild-type p53 [WT7 (Wilms), WT14 (atypical teratoid malignant rhabdoid tumor/kidney), BT41 (ependymoma), NB-1643 (neuroblastoma), and rhabdomyosarcomas (Rh10, Rh28, and Rh30); note that the heterozygous mutant genotype for Rh30 reported previously (35) was from a cell line, whereas Rh30 used here is the parent tumor derived directly from the patient sample and heterografted into mice was from a cell line, whereas Rh30 used here is the parent tumor derived directly from the patient sample and heterografted into mice were unresponsive to this agent. Neither does mutant p53 predict intrinsic resistance to depsipeptide; BT35 (single point mutation leading to a Gly-to-Asp transition at residue 244), an anaplastic astrocytoma line, was relatively responsive (6 of 16 tumors showing volume regression of ≥50%), whereas the other "resistant" tumors were either mutant (SKNEP and GBM2) or heterozygous (WT10).

In vitro, depsipeptide induced p21cip1 in several cell lines (30, 36). Consistent with the p53 genotype, depsipeptide induced p21cip1 in BT27 and IRS56 tumors. Therefore, the induction of p21cip1 in these tumors could lead to growth arrest as was apparent during the growth inhibitory studies. p21cip1 can induce G1 arrest by inhibiting the cyclin D–, cyclin E–, and cyclin A–dependent kinases (13). Espinosa and Emerson (37) used an elegant in vitro transcription assay to show that a preassembled chromatin template containing the p21WAF1/cip1 promoter and far upstream binding sites were transcriptionally silent when incubated in HeLa nuclear extract but that transcription could be strongly and synergistically activated by preincubation of the template with purified p53 and p300. Remarkably, they found that wild-type p53 and a mutant derivative in which Lys370, Lys372, Lys373, Lys380, and Lys382 changed to Arg (no acetylation by p300) displayed identical levels of activation. In addition, an increased accumulation of histone H4 was detected in the p21cip1 promoter and the structural gene (exon 2) on treatment with depsipeptide as assessed by chromatin immunoprecipitation assay. Thus, increased expression of p21cip1 due to depsipeptide is regulated, at least in part, by the degree of acetylation of the gene-associated histones (36). In tumors with mutant p53, no increased p21cip1 was detected.

Thus, our results in vivo suggest that factors other than p53 genotype affect the sensitivity to this HDACI. Efflux of depsipeptide by MDR1 and MRP1 transporters has been reported (38, 39). Although we have not directly examined the expression of ABC transporters in these tumor lines, depsipeptide sensitivity/resistance does not correlate with sensitivity to another MDR1 substrate, vincristine. For example, WT7, WT8, and WT10, Wilms' tumor lines,7 and Rh28 and Rh30 rhabdomyosarcoma xenografts (28) are exquisitely sensitive to vincristine regressing completely, whereas only WT8 was found sensitive to depsipeptide in the current study.

Xiao et al. (39) used gene expression changes to identify increased expression of caspase-9 and mitogen-activated protein kinase phosphatase-1 as markers of depsipeptide sensitivity. Limited microarray analysis (Affymetrix U133 2.0 Plus Chip) on BT27 tumor (CR) did not show any increase in the expression of caspase-9 or mitogen-activated protein kinase phosphatase-1 8 hours after depsipeptide treatment. Both caspase-8 and caspase-9 activate caspase-3, among other caspases (40), which in turn cleave several substrates, including the DNA repair enzyme PARP, into fragments of 87 and 28 kDa (41). Depsipeptide has been shown to activate caspase-8 and caspase-10, which in turn cleave caspase-3 and caspase-7, leading to apoptotic cell death in myeloid leukemia cell lines HL60 and K562 (42). The presence of degraded PARP-1 is generally considered as a marker of apoptosis (43). Two sensitive (BT27 and IRS56) and two resistant tumors (GBM2 and SKNEP) had an increase in cleaved PARP and therefore would suggest an increase in apoptosis 24 hours after depsipeptide treatment. However, the resistant tumors and IRS56 had cleaved PARP detectable in control tumors, suggesting that some apoptosis is occurring under normal conditions. Thus, drug-induced cleavage of PARP does not seem to correlate with tumor sensitivity to depsipeptide.

Recently, Kodani et al. (26) reported that A549 cells that were relatively sensitive to depsipeptide suppressed the phosphorylation of Akt at Ser473. In contrast, depsipeptide had no effect on the phosphorylation of Akt in PC14 cells that were resistant to depsipeptide treatment. Our results showed no consistent pattern of changes in Akt phosphorylation with depsipeptide sensitivity. Low to undetectable Akt phosphorylation (Ser473) in control BT27 (sensitive) and WT10 (resistant) was observed. The other tumors, irrespective of drug sensitivity, had high levels of phosphorylated Akt. Drug treatment either decreased pAkt levels (BT35, SKNEP) or caused an increase (BT27). Of interest is the dramatic increase at 48 hours in WT10 xenografts, which was consistent in each of the three tumors examined. Thus, there was no consistent change in pAkt that correlated with depsipeptide responsiveness. Therefore, decreased phosphorylation of Akt at Ser473 may not be a robust marker for tumors that will respond to depsipeptide therapy.

In summary, our study suggests a relatively low response rate to depsipeptide in a panel of pediatric tumor models at dose levels that in mice give systemic exposures similar to that achieved in patients. Our study shows that depsipeptide inhibits its target in vivo (i.e., causes increased histone acetylation), but this does not correlate with drug sensitivity. Although the magnitude and duration of acetylation may be an important factor in whether a tumor responds, as may be the p53 status of the tumor, we were unable to define molecular characteristics that predict in vivo tumor sensitivity. Importantly, our study forms the basis for determining the activity of depsipeptide or possibly other HDACIs when combined with conventional cytotoxic agents or agents directed at molecular targets pertinent to pediatric cancers.


   Acknowledgments
 
We thank the Hartwell Center for Bioinformatics and Biotechnology at St. Jude for sequencing p53.


   Footnotes
 
Grant support: USPHS awards CA23099, CA96696, and CA21765 (Cancer Center Support Grant) and American, Lebanese, Syrian, Associated Charities.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

5 In preparation. Back

6 C.F. Stewart, unpublished data. Back

7 P.J. Houghton, unpublished data. Back

Received 6/ 8/05; revised 10/11/05; accepted 10/20/05.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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