Abstract
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.
Keywords: Preclinical Testing, Developmental Therapeutics, Vorinostat
INTRODUCTION
Vorinostat (suberoylanilide hydroxamic acid or SAHA) is a potent histone deacetylase (HDAC) inhibitor that inhibits both class I and class II enzymes [1]. 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 [1]. 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 [9]. 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.
MATERIALS AND METHODS
In vitro
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 [12].
In vivo
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 [18]. 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 [13]. Responses were determined using three activity measures as previously described [13]. A detailed description of the analysis methods is included in the Supplemental Response Definitions.
Statistical Methods
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.
Drugs and Formulation
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.
RESULTS
In vitro 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.
Table I.
Activity of Vorinostat against Cell Lines in the PPTP in Vitro Panel
| Cell Line | Histology | IC50 (μM) | Median IC50 Ratio | IC90 (μM) |
|---|---|---|---|---|
| RD | Rhabdomyosarcoma | 6.06 | 0.24 | 51.75 |
| Rh41 | Rhabdomyosarcoma | 0.88 | 1.63 | 2.81 |
| Rh18 | Rhabdomyosarcoma | 9.77 | 0.15 | 21.60 |
| Rh30 | Rhabdomyosarcoma | 1.72 | 0.83 | 4.36 |
| BT-12 | Rhabdoid | 7.70 | 0.19 | 20.33 |
| CHLA-266 | Rhabdoid | 2.49 | 0.58 | 15.45 |
| TC-71 | Ewing sarcoma | 1.28 | 1.13 | 2.64 |
| CHLA-9 | Ewing sarcoma | 1.30 | 1.11 | 4.50 |
| CHLA-10 | Ewing sarcoma | 2.48 | 0.58 | 8.75 |
| CHLA-258 | Ewing sarcoma | 0.61 | 2.37 | 12.22 |
| GBM2 | Glioblastoma | 1.59 | 0.90 | 9.86 |
| NB-1643 | Neuroblastoma | 1.44 | 1.00 | 61.32 |
| NB-EBc1 | Neuroblastoma | 2.50 | 0.57 | 4.69 |
| CHLA-90 | Neuroblastoma | 2.81 | 0.51 | 20.90 |
| CHLA-136 | Neuroblastoma | 2.40 | 0.60 | 25.79 |
| NALM-6 | ALL | 1.78 | 0.81 | 4.97 |
| COG-LL-317 | ALL | 0.55 | 2.62 | 1.84 |
| RS4;11 | ALL | 0.62 | 2.33 | 5.16 |
| MOLT-4 | ALL | 0.68 | 2.11 | 2.42 |
| CCRF-CEM | ALL | 1.30 | 1.11 | 4.75 |
| Kasumi-1 | AML | 0.80 | 1.79 | 3.67 |
| Karpas-299 | ALCL | 0.48 | 2.97 | 2.12 |
| Ramos-RA1 | NHL | 1.16 | 1.24 | 2.71 |
| Median | 1.44 | 1.00 | 2.46 | |
| Minimum | 0.48 | 0.15 | 1.84 | |
| Maximum | 9.77 | 2.97 | 61.32 |
In vivo testing
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).
Table II.
Activity for Vorinostat against the PPTP in Vivo Panel
| Xenograft Line |
Histology | KM Estimate of Median Time to Event |
P-value | EFS T/C |
Median Final RTV |
Tumor Volume T/C |
P- value |
Overall Group Response |
T/C Volume Activity |
EFS Activity |
Response Activity |
|---|---|---|---|---|---|---|---|---|---|---|---|
| KT-10 | Wilms | 12.0 | 0.032 | 1.3 | >4 | 0.67 | 0.15 | PD1 | Low | Low | Low |
| KT-11 | Wilms | 17.4 | 0.004 | 1.9 | >4 | 0.34 | <0.001 | PD2 | Int | Low | Int |
| KT-13 | Wilms | 12.8 | 0.197 | 1.3 | >4 | 0.71 | 0.05 | PD1 | Low | Low | Low |
| SK-NEP-1 | Ewing | 11.6 | 0.034 | 1.7 | >4 | 0.61 | 0.03 | PD2 | Low | Low | Int |
| EW5 | Ewing | 13.8 | 0.044 | 1.1 | >4 | 1.02 | 0.50 | PD1 | Low | Low | Low |
| EW8 | Ewing | 15.3 | <0.001 | 1.6 | >4 | 0.57 | 0.00 | PD2 | Low | Low | Int |
| TC-71 | Ewing | 10.4 | 0.143 | 1.8 | >4 | 0.48 | 0.04 | PD2 | Low | Low | Int |
| CHLA258 | Ewing | 10.4 | 0.349 | 1.2 | >4 | 0.90 | 0.37 | PD1 | Low | Low | Low |
| Rh30 | ALV RMS | 20.3 | 0.611 | 1.0 | >4 | 0.84 | 0.97 | PD1 | Low | Low | Low |
| Rh30R | ALV RMS | 20.9 | 0.012 | 1.3 | >4 | 0.63 | 0.04 | PD1 | Low | Low | Low |
| Rh41 | ALV RMS | 19.1 | <0.001 | 1.6 | >4 | 0.56 | <0.001 | PD2 | Low | Low | Int |
| Rh18 | EMB RMS | 19.8 | 0.063 | 1.5 | >4 | 0.57 | 0.01 | PD1 | Low | Low | Low |
| Rh36 | EMB RMS | 24.4 | 0.005 | 1.4 | >4 | 0.89 | 0.36 | PD1 | Low | Low | Low |
| BT-28 | Medulloblastoma | 5.7 | 0.052 | 1.2 | >4 | 0.82 | 0.08 | PD1 | Low | Low | Low |
| BT-45 | Medulloblastoma | 16.6 | 0.026 | 1.3 | >4 | 0.84 | 0.10 | PD1 | Low | Low | Low |
| BT-46 | Medulloblastoma | 7.3 | 0.625 | 1.2 | >4 | 0.91 | 0.58 | PD1 | Low | Low | Low |
| BT-44 | Ependymoma | 26.1 | 0.357 | 0.7 | >4 | 1.61 | 0.02 | PD1 | Low | Low | Low |
| BT-39 | Glioblastoma | 10.0 | 0.918 | 1.0 | >4 | 1.04 | 0.74 | PD1 | Low | Low | Low |
| D645 | Glioblastoma | 8.6 | 0.891 | 1.0 | >4 | 1.03 | 0.53 | PD1 | Low | Low | Low |
| D456 | Glioblastoma | 6.0 | 0.872 | 1.1 | >4 | 0.90 | 0.66 | PD1 | Low | Low | Low |
| NB-SD | Neuroblastoma | > EP | 0.225 | > 1.5 | 3.0 | 1.18 | 1.00 | PD2 | Low | NE | Int |
| NB-1771 | Neuroblastoma | 35.2 | <0.001 | 1.4 | >4 | 0.31 | <0.001 | PD1 | Int | Low | Low |
| NB-1691 | Neuroblastoma | 10.7 | 0.076 | 1.1 | >4 | 0.85 | 0.06 | PD1 | Low | Low | Low |
| NB-EBc1 | Neuroblastoma | 10.4 | 0.012 | 1.6 | >4 | 0.69 | 0.02 | PD2 | Low | Low | Int |
| CHLA-79 | Neuroblastoma | 27.5 | 0.041 | 1.1 | >4 | 0.78 | 0.48 | PD1 | Low | Low | Low |
| NB-1643 | Neuroblastoma | 12.9 | 0.021 | 1.3 | >4 | 0.48 | 0.00 | PD1 | Low | Low | Low |
| OS-1 | Osteosarcoma | 34.5 | 0.062 | 1.1 | >4 | 0.77 | 0.10 | PD1 | Low | Low | Low |
| OS-17 | Osteosarcoma | 24.2 | 0.004 | 1.8 | >4 | 0.49 | <0.001 | PD2 | Low | Low | Int |
| OS-9 | Osteosarcoma | > EP | 0.002 | > 1.6 | 3.2 | 0.67 | 0.03 | PD2 | Low | NE | Int |
| OS-31 | Osteosarcoma | 21.1 | 0.003 | 1.3 | >4 | 0.58 | 0.01 | PD1 | Low | Low | Low |
| ALL-2 | ALL B-precursor | 9.8 | 0.269 | 0.8 | >25 | . | PD1 | Low | Low | ||
| ALL-3 | ALL B-precursor | 8.2 | 0.396 | 0.8 | >25 | . | PD1 | Low | Low | ||
| ALL-4 | ALL B-precursor | 2.3 | 0.731 | 1.3 | >25 | . | PD1 | Low | Low | ||
| ALL-7 | ALL B-precursor | 2.0 | 1.000 | 1.0 | >25 | . | PD1 | Low | Low | ||
| ALL-8 | ALL T-cell | 6.7 | 0.519 | 1.1 | >25 | . | PD1 | Low | Low | ||
| ALL-16 | ALL T-cell | 15.8 | 0.072 | 1.4 | >25 | . | PD1 | Low | Low | ||
| ALL-17 | ALL B-precursor | 2.0 | 1.000 | 1.0 | >25 | . | PD1 | Low | Low | ||
| ALL-19 | All B-precursor | 1.0 | 0.674 | 1.0 | >25 | . | PD1 | Low | Low |
DISCUSSION
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 [9]. Major adverse events for vorinostat administered orally or intravenously were fatigue, diarrhea, anorexia, dehydration, myelosuppression and thrombocytopenia [10].
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[14]. 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.
Supplementary Material
Acknowledgments
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.
Reference List
- 1.Glaser KB. HDAC inhibitors: Clinical update and mechanism-based potential. Biochem Pharmacol. 2007 doi: 10.1016/j.bcp.2007.04.007. [DOI] [PubMed] [Google Scholar]
- 2.Richon VM. Cancer biology: mechanism of antitumour action of vorinostat (suberoylanilide hydroxamic acid), a novel histone deacetylase inhibitor. British journal of cancer. 2006;95:5. [Google Scholar]
- 3.Richon VM, Sandhoff TW, Rifkind RA, et al. Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation. Proceedings of the National Academy of Sciences of the United States of America. 2000;97(18):10014–10019. doi: 10.1073/pnas.180316197. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Piekarz RL, Robey R, Sandor V, et al. Inhibitor of histone deacetylation, depsipeptide ( FR901228), in the treatment of peripheral and cutaneous T-cell lymphoma: a case report. Blood. 2001;98(9):2865–2868. doi: 10.1182/blood.v98.9.2865. [DOI] [PubMed] [Google Scholar]
- 5.Prince HM, George D, Patnaik A, et al. Phase I study of oral LBH589, a novel deacetylase (DAC) inhibitor in advanced solid tumors and non-Hodgkin’s lymphoma. J Clin Oncol. 2007;25(18S) Abstr #3500. [Google Scholar]
- 6.Duvic M, Talpur R, Ni X, et al. Phase 2 trial of oral vorinostat (suberoylanilide hydroxamic acid, SAHA) for refractory cutaneous T-cell lymphoma (CTCL) Blood. 2007;109(1):31–39. doi: 10.1182/blood-2006-06-025999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Duvic M, Vu J. Vorinostat: a new oral histone deacetylase inhibitor approved for cutaneous T-cell lymphoma. Expert opinion on investigational drugs. 2007;16(7):1111–1120. doi: 10.1517/13543784.16.7.1111. [DOI] [PubMed] [Google Scholar]
- 8.Olsen EA, Kim YH, Kuzel TM, et al. Phase IIb multicenter trial of vorinostat in patients with persistent, progressive, or treatment refractory cutaneous T-cell lymphoma. J Clin Oncol. 2007;25(21):3109–3115. doi: 10.1200/JCO.2006.10.2434. [DOI] [PubMed] [Google Scholar]
- 9.Mann BS, Johnson JR, He K, et al. Vorinostat for treatment of cutaneous manifestations of advanced primary cutaneous T-cell lymphoma. Clin Cancer Res. 2007;13(8):2318–2322. doi: 10.1158/1078-0432.CCR-06-2672. [DOI] [PubMed] [Google Scholar]
- 10.Kelly WK, O’Connor OA, Krug LM, et al. Phase I study of an oral histone deacetylase inhibitor, suberoylanilide hydroxamic acid, in patients with advanced cancer. J Clin Oncol. 2005;23(17):3923–3931. doi: 10.1200/JCO.2005.14.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Kelly WK, Richon VM, O’Connor O, et al. Phase I clinical trial of histone deacetylase inhibitor: suberoylanilide hydroxamic acid administered intravenously. Clin Cancer Res. 2003;9(10 Pt 1):3578–3588. [PubMed] [Google Scholar]
- 12.Houghton PJ, Morton CL, Kolb EA, et al. Initial testing (stage 1) of the proteasome inhibitor bortezomib by the pediatric preclinical testing program. Pediatr Blood Cancer. 2007 doi: 10.1002/pbc.21214. [DOI] [PubMed] [Google Scholar]
- 13.Houghton PJ, Morton CL, Tucker C, et al. The pediatric preclinical testing program: Description of models and early testing results. Pediatr Blood Cancer. 2006 doi: 10.1002/pbc.21078. [DOI] [PubMed] [Google Scholar]
- 14.Keshelava N, Davicioni E, Wan Z, et al. Histone deacetylase 1 gene expression and sensitization of multidrug-resistant neuroblastoma cell lines to cytotoxic agents by depsipeptide. Journal of the National Cancer Institute. 2007;99(14):1107–1119. doi: 10.1093/jnci/djm044. [DOI] [PubMed] [Google Scholar]
- 15.Thiele CJ, Reynolds CP, Israel MA. Decreased expression of N-myc precedes retinoic acid-induced morphological differentiation of human neuroblastoma. Nature. 1985;313(6001):404–406. doi: 10.1038/313404a0. [DOI] [PubMed] [Google Scholar]
- 16.Reynolds CP, Matthay KK, Villablanca JG, et al. Retinoid therapy of high-risk neuroblastoma. Cancer letters. 2003;197(1–2):185–192. doi: 10.1016/s0304-3835(03)00108-3. [DOI] [PubMed] [Google Scholar]
- 17.Coffey DC, Kutko MC, Glick RD, et al. Histone deacetylase inhibitors and retinoic acids inhibit growth of human neuroblastoma in vitro. Medical and pediatric oncology. 2000;35(6):577–581. doi: 10.1002/1096-911x(20001201)35:6<577::aid-mpo18>3.0.co;2-3. [DOI] [PubMed] [Google Scholar]
- 18.Kutko MC, Glick RD, Butler LM, et al. Histone deacetylase inhibitors induce growth suppression and cell death in human rhabdomyosarcoma in vitro. Clin Cancer Res. 2003;9(15):5749–5755. [PubMed] [Google Scholar]
- 19.Hallahan AR, Pritchard JI, Chandraratna RA, et al. BMP-2 mediates retinoid-induced apoptosis in medulloblastoma cells through a paracrine effect. Nat Med. 2003;9(8):1033–1038. doi: 10.1038/nm904. [DOI] [PubMed] [Google Scholar]
- 20.Gumireddy K, Sutton LN, Phillips PC, et al. All-trans-retinoic acid-induced apoptosis in human medulloblastoma: activation of caspase-3/poly(ADP-ribose) polymerase 1 pathway. Clin Cancer Res. 2003;9(11):4052–4059. [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.