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Vorinostat, a histone deacetylase inhibitor, was evaluated against the in vitro and in vivo childhood solid tumor and leukemia models in the Pediatric Preclinical Testing Program (PPTP). In vitro testing was performed by the DIMSCAN cytotoxicity assay. In vivo, vorinostat was administered intraperitoneally to mice bearing xenografts. Vorinostat demonstrated 2-log cell growth inhibitory activity in vitro, but generally at concentrations not sustainable in the clinic. No objective responses were observed for any of the solid tumor or acute lymphoblastic leukemia xenografts. Preclinical studies with appropriate drug combinations may provide direction for further clinical evaluations of vorinostat against selected pediatric cancers.
Vorinostat (suberoylanilide hydroxamic acid or SAHA) is a potent histone deacetylase (HDAC) inhibitor that inhibits both class I and class II enzymes . The former group includes HDACs 1, 2, 3 and 8 and are primarily localized to the nucleus, while the latter group includes HDACs 4, 5, 6, 7, 9, and 10 and are primarily found in the cytoplasm . HDAC inhibitors induce hyperacetylation of a number of proteins, resulting in a plethora of downstream effects. Increased levels of acetylated histones generally result in a more open chromatin conformation that is associated with active gene expression [2,3]. HDAC inhibitors also impair mitotic progression, producing defects in chromosome condensation, segregation, and kinetochore assembly [1,2]. Non-histone proteins that show increased acetylation in response to HDAC inhibition include numerous transcription factors (e.g., E2F-1, NF-kappaB, GATA-1, Bcl-6, etc.), Hsp90, Ku70, and α-tubulin [1,2]. Not yet understood is which of these biological effects of HDAC inhibitors is primarily associated with anticancer activity [1,2].
Vorinostat induces differentiation, growth arrest, or apoptosis in a broad range of cancer cell lines [2–4]. It has also demonstrated anti-tumor activity in a number of in vivo models including leukemia, lymphoma, prostate, and breast cancer [2,5–7]. Like other HDAC inhibitors under clinical evaluation [4,5], vorinostat’s greatest clinical activity has been against cutaneous T-cell lymphoma [6–8]. Vorinostat was approved by FDA in 2006 for the treatment of cutaneous manifestations of advanced cutaneous T-cell lymphoma . Objective responses were also reported in vorinostat phase 1 studies for patients with mesothelioma, thyroid cancer, laryngeal cancer, Hodgkin disease, and diffuse large B-cell lymphoma [10,11]. Vorinostat has entered phase 1 evaluation in children with cancer.
Cell sensitivity was determined using DIMSCAN. Cells were incubated in the presence of vorinostat for 96 hours at concentrations from 0.01 μM to 100 μM and analyzed as previously described .
CB17SC-M scid−/− female mice (Taconic Farms, Germantown NY), were used to propagate subcutaneously implanted kidney/rhabdoid tumors, sarcomas (Ewing, osteosarcoma, rhabdomyosarcoma), neuroblastoma, and non-glioblastoma brain tumors, while BALB/c nu/nu mice were used for glioma models, as previously described [15–17]. Human leukemia cells were propagated by intravenous inoculation in female non-obese diabetic (NOD)/scid −/− mice as described previously . All mice were maintained under barrier conditions and experiments were conducted using protocols and conditions approved by the institutional animal care and use committee of the appropriate consortium member. Tumor volumes (solid tumor xenografts) or percentages of human CD45-positive (hCD45) cells (ALL xenografts) were determined as previously described . Responses were determined using three activity measures as previously described . A detailed description of the analysis methods is included in the Supplemental Response Definitions.
The exact log-rank test, as implemented using Proc StatXact for SAS®, was used to compare event-free survival distributions between treatment and control groups. P-values were two-sided and were not adjusted for multiple comparisons given the exploratory nature of the studies.
Vorinostat was provided to the Pediatric Preclinical Testing Program by Merck through the Cancer Therapy Evaluation Program (NCI). For in vivo testing vorinostat was dissolved in DMSO (final concentration 10%) and diluted in PEG400 (final concentration 45%) in water and administered intraperitoneally daily × 5 for 6 weeks at a dose of 125 mg/kg. Vorinostat was provided to each consortium investigator in coded vials for blinded testing.
Vorinostat was uniformly able to inhibit growth of the cell lines from the PPTP in vitro panel (Table I). The median IC50 for the entire panel was 1.44 μM with a range of 0.48 μM to 9.77 μM. The median IC90 for the panel was 4.86 μM with a range of 1.84 to 61.32 μM.
Vorinostat was evaluated in 45 xenograft models. Eight of 424 (1.9%) mice died in the vehicle control arm and 53 of 431 (12.3%) in the vorinostat treatment arm. Seven lines (BT-29, KT-14, Rh28, BT-36, GBM2, OS-2, and OS-33) were excluded from analysis due to toxicity greater than 25 percent. A complete summary of results is provided in Supplemental Table I, including total numbers of mice, number of mice that died (or were otherwise excluded), numbers of mice with events and average times to event, tumor growth delay, as well as numbers of responses and T/C values. No objective responses were observed in any of the models. The best responses observed were nine examples of PD2 (progressive disease with growth delay). These included TC-71 (Ewing), NB-Ebc1 (neuroblastoma) and Rh41 (rhabdomyosarcoma) xenografts that were also tested in vitro and whose IC90 values were the lowest in the Ewing, neuroblastoma, or rhabdomyosarcoma cell line panels. Vorinostat induced significant differences in EFS distribution compared to controls in 16 of 38 evaluable xenografts (Table II), although no xenografts met the criteria for intermediate or high activity (EFS T/C value > 2.0 and a significant difference in EFS distribution).
Phase I clinical trials of vorinostat demonstrated that the agent can be administered safely for a prolonged period of time at doses that inhibit HDAC activity . Major adverse events for vorinostat administered orally or intravenously were fatigue, diarrhea, anorexia, dehydration, myelosuppression and thrombocytopenia .
The antitumor activity of vorinostat was studied in the PPTP in vitro and in vivo panels, to determine if this agent had significant activity against pediatric leukemias or solid tumors. Vorinostat achieved cell growth inhibition in all tested cell lines in vitro; IC50 values ranged from 0.48 to 9.8 μM. This range is in agreement with growth inhibitory effect of vorinostat demonstrated in other in vitro models [2–4]. However, drug concentrations that resulted in 1 log inhibition (IC90) were generally beyond the clinically achievable levels (1–2 μM).
For in vivo testing, vorinostat was administered at the dose and schedule that has previously been shown to induce acetylation of histones H3 and H4, pharmacodynamic markers of HDAC inhibition [2,4]. Vorinostat induced differences in EFS distribution in solid tumor xenografts, but no objective responses were observed in either the ALL or the solid tumor panels. Thus, vorinostat as a single agent did not show significant anti-tumor activity in either the in vitro or in vivo panels.
In spite of the lack of activity by single agent vorinostat in the PPTP models, it does remain an anti-tumor agent of interest for use in drug combinations. For example, HDAC1 overexpression has recently been shown to be one mechanism of multi-drug resistance in neuroblastoma cell lines that can be reversed with HDAC inhibition. Combinations of HDAC inhibitors with retinoids are also of interest in the pediatric setting based on previously published work for the activity of retinoids as single agents against certain diagnoses (e.g., for neuroblastoma and medulloblastoma) and based on the activity described for combinations of retinoids and HDAC inhibitors [15–20]. Thus, while vorinostat did not show promising single agent activity in PPTP testing, preclinical studies with appropriate drug combinations may provide direction for further clinical evaluations of vorinostat against selected pediatric cancers.
This work was supported by NO1-CM-42216, CA21765, and CA108786 from the National Cancer Institute and used vorinostat provided by Merck. In addition to the authors, the report represents work contributed by the following: Sherry Ansher, Catherine A. Billups, Joshua Courtright, Edward Favours, Henry S. Friedman, Debbie Payne-Turner, Charles Stopford, Mayamin Tajbakhsh, Chandra Tucker, Joe Zeidner, Ellen Zhang, and Jian Zhang. Children’s Cancer Institute Australia for Medical Research is affiliated with the University of New South Wales and Sydney Children’s Hospital.