efficacy of class i and ii vs class iii histone deacetylase inhibitors in neuroblastoma

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Efficacy of class I and II vs class III histone deacetylase inhibitors in neuroblastoma Timothy B. Lautz a,b , Jessica A. Naiditch a,b , Sandra Clark b , Fei Chu b, , Mary Beth Madonna a,b, a Department of Surgery, Children's Memorial Hospital, Northwestern University, Chicago, IL 60614, USA b Cancer Biology and Epigenetics Program, Children's Memorial Research Center, Northwestern University, Chicago, IL 60614, USA Received 3 March 2012; accepted 6 March 2012 Key words: Histone deacetylase inhibitor; Vorinostat; Neuroblastoma; Chemotherapy resistance Abstract Background: Histone deacetylase (HDAC) inhibitors have shown promise in the treatment of resistant and refractory tumors including neuroblastoma. The goal of the study was to compare the efficacy of a class III HDAC inhibitor (cambinol) to a class I and II inhibitor (vorinostat). Methods: In vitro efficacy of vorinostat and cambinol, alone or in combination with doxorubicin, was assessed by 2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide calorimetric assay using both wild-type (WT) and doxorubicin-resistant (DoxR) SK-N-SH neuroblastoma cells. In vivo efficacy was determined using the same drug combinations in nude mice bearing xenograft implants of WT and DoxR cells on opposite flanks. Results: Vorinostat and cambinol were efficacious against WT and DoxR neuroblastoma cells in vitro. In WT cells, the potency of the doxorubicin itself overshadowed any effect of cotherapy with vorinostat or cambinol. The effect of vorinostat and/or cambinol on the DoxR cells was constant across progressively increasing doses of doxorubicin. In the in vivo model, the efficacy of doxorubicin itself (88% reduction in tumor volume) again overshadowed any effect of cotreatment with vorinostat or cambinol on the WT tumors. However, in the DoxR tumors, doxorubicin alone had no efficacy, but cotreatment with either cambinol or vorinostat suppressed tumor growth (70% and 91% reduction in tumor volume, respectively). Conclusions: Both the class III HDAC inhibitor cambinol and the class I/II HDAC inhibitor vorinostat have efficacy against SK-N-SH neuroblastoma cells, including those resistant to doxorubicin. © 2012 Elsevier Inc. All rights reserved. Neuroblastoma is the most common extracranial solid tumor of childhood and accounts for 15% of cancer-related deaths in children. The natural history of neuroblastoma and its responsiveness to standard treatment regimens are highly variable. Survival in infants is excellent, and tumors in the youngest children often regress without therapy [1]. In contrast, older children with neuroblastoma, especially those with unfavorable histology and metastatic disease, have a poor prognosis. Survival in this population remains only 20% to 35% despite intensive chemotherapy and autologous There are no disclaimers or funding sources to report. Corresponding authors. 2300 Children's Plaza, Box 63, Chicago, IL 60614, USA. Tel.: +1 773 880 4912; fax: +1 773 880 4588. E-mail addresses: [email protected] (M.B. Madonna), [email protected] (F. Chu). www.elsevier.com/locate/jpedsurg 0022-3468/$ see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2012.03.039 Journal of Pediatric Surgery (2012) 47, 12671271

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Page 1: Efficacy of class I and II vs class III histone deacetylase inhibitors in neuroblastoma

www.elsevier.com/locate/jpedsurg

Journal of Pediatric Surgery (2012) 47, 1267–1271

Efficacy of class I and II vs class III histone deacetylaseinhibitors in neuroblastoma☆

Timothy B. Lautz a,b, Jessica A. Naiditch a,b, Sandra Clark b,Fei Chub,⁎, Mary Beth Madonna a,b,⁎

aDepartment of Surgery, Children's Memorial Hospital, Northwestern University, Chicago, IL 60614, USAbCancer Biology and Epigenetics Program, Children's Memorial Research Center, Northwestern University, Chicago,IL 60614, USA

Received 3 March 2012; accepted 6 March 2012

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Key words:Histone deacetylaseinhibitor;

Vorinostat;Neuroblastoma;Chemotherapy resistance

AbstractBackground: Histone deacetylase (HDAC) inhibitors have shown promise in the treatment of resistantand refractory tumors including neuroblastoma. The goal of the study was to compare the efficacy of aclass III HDAC inhibitor (cambinol) to a class I and II inhibitor (vorinostat).Methods: In vitro efficacy of vorinostat and cambinol, alone or in combination with doxorubicin, wasassessed by 2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide calorimetric assay using bothwild-type (WT) and doxorubicin-resistant (DoxR) SK-N-SH neuroblastoma cells. In vivo efficacy wasdetermined using the same drug combinations in nude mice bearing xenograft implants of WT andDoxR cells on opposite flanks.Results: Vorinostat and cambinol were efficacious against WT and DoxR neuroblastoma cells in vitro. InWT cells, the potency of the doxorubicin itself overshadowed any effect of cotherapy with vorinostat orcambinol. The effect of vorinostat and/or cambinol on the DoxR cells was constant across progressivelyincreasing doses of doxorubicin. In the in vivo model, the efficacy of doxorubicin itself (88% reduction intumor volume) again overshadowed any effect of cotreatment with vorinostat or cambinol on the WTtumors. However, in the DoxR tumors, doxorubicin alone had no efficacy, but cotreatment with eithercambinol or vorinostat suppressed tumor growth (70% and 91% reduction in tumor volume, respectively).Conclusions: Both the class III HDAC inhibitor cambinol and the class I/II HDAC inhibitor vorinostathave efficacy against SK-N-SH neuroblastoma cells, including those resistant to doxorubicin.© 2012 Elsevier Inc. All rights reserved.

Neuroblastoma is the most common extracranial solidtumor of childhood and accounts for 15% of cancer-related

☆ There are no disclaimers or funding sources to report.⁎ Corresponding authors. 2300 Children's Plaza, Box 63, Chicago,60614, USA. Tel.: +1 773 880 4912; fax: +1 773 880 4588.E-mail addresses: [email protected] (M.B. Madonna),

[email protected] (F. Chu).

022-3468/$ – see front matter © 2012 Elsevier Inc. All rights reserved.oi:10.1016/j.jpedsurg.2012.03.039

deaths in children. The natural history of neuroblastoma andits responsiveness to standard treatment regimens are highlyvariable. Survival in infants is excellent, and tumors in theyoungest children often regress without therapy [1]. Incontrast, older children with neuroblastoma, especially thosewith unfavorable histology and metastatic disease, have apoor prognosis. Survival in this population remains only20% to 35% despite intensive chemotherapy and autologous

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1268 T.B. Lautz et al.

bone marrow transplant [2]. Acquired resistance to chemo-therapeutic agents remains a primary barrier to successfultreatment of neuroblastoma and drives the search foralternative therapeutic agents to treat children with relapsedand resistant disease.

Histone deacetylase (HDAC) inhibitors are a novel classof drugs that have demonstrated promise in the treatmentof a wide variety of pediatric malignancies. The HDACfamily consists of 18 members classified into 4 groups(classes I-IV) based on their sequence homologies [3].Histone deacetylases are important regulators of genetranscription via their role in deacetylating the amino-terminal tails of histones. In addition, epigenetic modifi-cation of a growing list of nonhistone HDAC substrates hasbeen implicated in various aspects of cancer physiologyand tumorigenesis [3-5]. Histone deacetylase inhibitorshave demonstrated a wide range of effects on cancer cells,including growth inhibition, induction of cell death,differentiation, and antiangiogenesis [4,6,7]. Vorinostat isthe first Food and Drug Administration–approved HDACinhibitor and is currently in phase I trials for a number ofhematologic and solid organ malignancies [8]. Histonedeacetylase inhibitors have shown therapeutic promiseagainst neuroblastoma in preclinical studies, and vorinostathas been well tolerated in early clinical studies of childrenwith recurrent solid tumors [9-11].

Vorinostat broadly inhibits class I and II but not class IIIHDACs. The class III HDACs (sirtuins 1–7), which areevolutionarily distinct and depend on a nicotinamide adeninedinucleotide cofactor, can be inhibited by cambinol [12]. Theclass III HDAC sirt1 has been shown to regulate themultidrug resistance molecule P-glycoprotein in neuroblas-toma and may, therefore, represent a target for treating drug-resistant tumors [13].

The aim of the present study was to compare the relativeefficacy of the class I and II HDAC inhibitor vorinostat withthat of the class III HDAC inhibitor cambinol.

1. Methods

1.1. Cell lines and reagents

Human neuroblastoma (SK-N-SH) cells were purchasedfrom American Type Culture Collection (Manassas, VA).Doxorubicin-resistant (DoxR) cells were generated byincubating the wild-type (WT) cells with incrementalconcentrations of doxorubicin (Sigma, St Louis, MO)ranging from 10−9 to 10−6 M during a 9-month period.Doxorubicin resistance was confirmed by 2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) calorimetric assay as previously described [14].Dulbecco modified Eagle medium and fetal bovine serum fortissue culture were obtained from BioWhittaker (Walkers-ville, MD).

1.2. In vitro drug efficacy

The effect of treatment with vorinostat, cambinol, and/ordoxorubicin on cell proliferation was assessed by MTTcalorimetric assay. Cells were seeded in 96-well plates andincubated with logarithmic concentrations of doxorubicin(10−9-10−5 M), vorinostat (10−9-10−5 M), or cambinol(10−8-10−4 M) for 96 hours. Ten microliters of 5 mg/mLMTT solution was added to each well of the titration plateand incubated for 4 hours at 37°C. The cells were thensolubilized by the addition of 100 μL of 10% sodiumdodecyl sulfate/0.01 mmol/L HCl and incubated for 15 hoursat 37°C. The absorbance of each well was determined in anenzyme-linked immunosorbent assay–plated reader using anactivation wavelength of 570 nm and a reference wavelengthof 650 nm. Relative cell proliferation was determined bycomparison with untreated control cells.

We then assessed for a synergistic effect of cotreatment ofvorinostat and/or cambinol with doxorubicin. The concen-tration that reduced the number of viable cells by 25% (IC25)of cambinol and vorinostat was determined from thepreviously described MTT assays. Cell proliferation wasthen assessed by MTT assay using cells cotreated with theIC25 dose of cambinol and/or vorinostat combined withlogarithmic concentrations of doxorubicin (10−9-10−5 M).

1.3. In vivo drug efficacy

The in vivo efficacy of cambinol and vorinostat treatmentwas assessed in a nude mouse model. The animal protocolwas approved by the Animal Care and Use Committee of theChildren's Memorial Research Center (#2010-15). Nudemice (strain CD1; Charles River Laboratories, Wilmington,MA) of 5 to 6 weeks of age and weighing approximately 30 greceived subcutaneous tumor injections. Wild-type andDoxR SK-N-SH cells (106 cells in 100 μL) were implantedinto opposite flanks of 42 nude mice. When tumors werepalpable (∼50 mm3), the mice were divided into 7 groups of6 mice each, as follows: (a) control, (b) vorinostat (50 mg/kgper dose) alone, (c) cambinol (100 mg/kg per dose) alone, (d)doxorubicin (2.5 mg/kg per dose) alone, (e) vorinostat plusdoxorubicin, (f) cambinol plus doxorubicin, and (g) vorino-stat plus cambinol plus doxorubicin. A total of 3 injectionswere given during a 1-week period. Tumor measurementswere obtained biweekly and converted to tumor volumeusing the equation length × (width/2)2. Weights wereobtained weekly. The mice were humanely killed when themaximal dimension of either tumor reached 15 mm.

1.4. Statistical analysis

Continuous variables are reported as a mean ±standard error, and groups are compared by Studentt test. All analyses were 2 tailed, and a P b .05 wasconsidered significant.

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2. Results

2.1. In vitro drug efficacy

The effect of single therapy with cambinol, vorinostat,or doxorubicin on cell proliferation in WT and DoxR SK-N-SH neuroblastoma cells was assessed using the MTTcalorimetric assay. As expected, the WT cells were veryresponsive to doxorubicin (IC50 = 2.4 × 10−7 M), whereasthe DoxR cells were highly resistant to doxorubicin therapy(IC50 = 4.8 × 10−5 M). Doxorubicin-resistance did not,however, cause cross-resistance to HDAC inhibitor therapy.The WT and DoxR cells were equally responsive to bothcambinol (IC50 = 3.5 × 10−5 and 1.8 × 10−5 M,respectively) and vorinostat (IC50 = 3.4 × 10−6 and 3.7 ×10−6 M, respectively).

Cells were then cotreated using doxorubicin withcambinol and/or vorinostat to assess for a synergistic effectand to determine if HDAC inhibition could restoredoxorubicin sensitivity. Wild-type and DoxR cells receivedtreatment at the approximate IC25 dose with cambinol (30μM) and/or vorinostat (1.5 μM) combined with logarithmicdoses of doxorubicin (10−9-10−5 M). Fig. 1 shows theefficacy of these treatment combinations in (a) WT and (b)

Fig. 1 In vitro efficacy of doxorubicin cotreatment with cambinol(30 μM) and/or vorinostat (1.5 μM) in WT (A) and DoxR (B) SK-N-SH neuroblastoma cells as assessed by MTT calorimetric assay.

DoxR cell lines. As designed, both cambinol and vorinostattherapies reduced WT cell proliferation by approximately25% at low or no doses of doxorubicin. With higher doses ofdoxorubicin, the effect of the doxorubicin therapy dwarfedany effect of the HDAC inhibitors. As such, the IC50 dose ofdoxorubicin (2.4 × 10−7 M) was not significantly altered bycotreatment with vorinostat (2.0 × 10−7 M), cambinol (2.3 ×10−7 M), or both (2.8 × 10−7 M).

Treatment with cambinol and/or vorinostat reducedproliferation of DoxR cells (Fig. 1B) but did not changethe shape of the dose-response curve for doxorubicin. Inother words, the effect of cambinol and/or vorinostattreatment was constant across increasing doses of doxoru-bicin cotherapy. However, because of the baselinereduction in cell proliferation, cotreatment with cambinol,vorinostat, or both reduced the IC50 dose for doxorubicin(3.6 × 10−5, 1.1 × 10−5, and 6.6 × 10−6 M, respectively)compared with treatment with doxorubicin alone (4.8 ×10−5 M).

2.2. In vivo drug efficacy

The in vivo efficacy of the above drug combinations wasassessed in nude mice bearing WT and DoxR xenografttumor implants on opposite flanks. Fig. 2 (A, B) shows theWT and DoxR tumor volume over time among mice in thefollowing treatment groups: (1) control, (2) vorinostat, (3)cambinol, (4) doxorubicin, (5) vorinostat and doxorubicin,(6) cambinol and doxorubicin, and (7) vorinostat, cambinol,and doxorubicin. The effect of the different treatmentregimens on tumor volume, relative to the control, at 18days after the first injection is quantified in Fig. 2C. In theWT tumors, the efficacy of doxorubicin (88% reduction intumor volume, P = .006) overshadowed any effect ofcotreatment with vorinostat (7% reduction, P = .26) orcambinol (48% reduction, P = .10). However, in the DoxRtumors, doxorubicin alone had no efficacy. Treatment witheither cambinol or vorinostat suppressed tumor growth(63% [P = .07] and 48% [P = .10] reduction, respectively).Furthermore, mice cotreated with cambinol plus doxorubi-cin or vorinostat plus doxorubicin had an even greaterreduction in tumor volume (91% [P = .005] and 70% [P =.06], respectively).

Monotherapy or dual therapy with combinations of theabove agents was well tolerated. The mean weight 3 weeksafter treatment initiation ranged from 95% to 131% of thestarting weight in these groups. Fig. 2D shows the survivalof the mice by treatment group. Only 4 mice receivingmonotherapy or dual therapy died of drug lethality (1 in thecambinol group, 1 in the doxorubicin group, 1 in thevorinostat plus doxorubicin group, and 1 in the cambinolplus doxorubicin group). The remaining mice werehumanely killed when the tumor on either flank reached15 mm in maximal dimension. In contrast, the triplecombination of doxorubicin, vorinostat, and cambinol

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Fig. 2 In vivo efficacy of cambinol, vorinostat, and doxorubicin combinatorial therapies in a nude mouse xenograft model. The mice weredivided into 7 groups of 6 animals after tumors were palpable, and treatments were given as 3 injections for 8 days, as follows: (1) control, (2)vorinostat, (3) cambinol, (4) doxorubicin, (5) vorinostat and doxorubicin, (6) cambinol and doxorubicin, and (7) vorinostat, cambinol, anddoxorubicin. Tumor volumes over time for the WT (A) and DoxR (B) xenografts are shown graphically. C, Tumor volumes at 18 days after thefirst injection as a fraction of the control group (mean ± SEM); ⁎a statistically significant reduction in tumor volume compared with the control(P b .05). Triple therapy with vorinostat, cambinol, and doxorubicin caused excessive toxicity preventing the assessment of tumor volume inthat group. D, The survival of the mice in all treatment groups. All regimens except the triple therapy were well tolerated.

1270 T.B. Lautz et al.

caused excessive toxicity resulting in rapid weight loss andthe death of 3 of 6 mice by 11 days and 5 of 6 by 14 daysafter the first injection.

3. Discussion

The class I and II HDAC inhibitor vorinostat has garneredmuch recent attention as a potential adjunct therapy forchildren with relapsed and resistant neuroblastoma. Vorino-stat has been or is being investigated in at least 4 phase I trialsfor this disease [8]. We herein confirm that vorinostat showsboth in vitro and in vivo efficacy against doxorubicin-sensitive and DoxR neuroblastoma cells. We furtherdemonstrate that the class III HDAC inhibitor cambinolappears at least as efficacious against neuroblastoma asvorinostat, although higher drug doses were required.Combined therapy with vorinostat and cambinol did nothave a significant synergistic effect in vitro and causedexcessive toxicity when given as triple therapy withdoxorubicin at these doses.

Class I and II HDAC inhibitors including vorinostat haveshown promise against neuroblastoma in several preclinicalstudies [15]. Vorinostat therapy, alone or in combination withother anticancer agents, has been shown to induce mitotic celldeath among drug-resistant neuroblastoma cells with p53 lossof function [16], to sensitize neuroblastoma cells to tumornecrosis factor-related apoptosis-inducing ligand-inducedapoptosis [17], and to potentiate the antineoplastic effect ofradiation therapy via down-regulation of a DNA-repairenzyme [18]. Likewise, the structurally related HDACinhibitor m-carboxycinnamic acid bis-hydroxamide has pre-viously demonstrated efficacy alone or in combinationwith all-trans-retinoic acid in a neuroblastoma xenograft model [9].Vorinostat was well tolerated in a phase I pediatric trial, and1 child with neuroblastoma even experienced a completeresponse [10]. There is ongoing national and internationalinterest in studying the safety and efficacy of this drug inchildren with high-risk and recurrent neuroblastoma.

Although most preclinical and clinical studies of HDACinhibitors in neuroblastoma have focused on vorinostat andrelated class I and II inhibitors, there is some evidence that theclass III sirtuin family of HDACs may also play a key role in

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neuroblastoma drug resistance. Previous work from ourlaboratory demonstrated that sirt1 is up-regulated in drug-resistant SK-N-SH neuroblastoma cell lines [13]. Smallinterfering RNA–mediated knockdown of sirt1 resulted indecreased expression of the multidrug resistance molecule P-glycoprotein and increased sensitivity to doxorubicin. Cambi-nol, a small molecular inhibitor of the class III HDACs(sirtuins), demonstrated efficacy against Burkitt cell lymphomaby inducing cell-cycle arrest andwaswell tolerated inmice [12].Cambinol has not been heretofore studied in neuroblastoma.

The current study offers insight into several key questionsregarding the use of HDAC inhibitors in neuroblastoma andprovides direction for future studies. We are the first todemonstrate that class III HDAC inhibition with cambinolmay be at least as efficacious as class I and II HDACinhibition with vorinostat and warrants further preclinicalstudy. We also show that vorinostat and cambinol areeffective in doxorubicin-sensitive and DoxR cells but thatneither drug has a major synergistic effect with doxorubicin.Histone deacetylase inhibitors do not appear to restoredoxorubicin sensitivity in the resistant lines. Further studiesare needed to determine if triple therapy with vorinostat,cambinol, and doxorubicin using reduced doses can betolerated without the toxicity seen herein and whether thiscombination has any therapeutic advantage. If additionalpreclinical studies support the efficacy of cambinol, futureclinical studies comparing the efficacy of class I/II vs class IIIHDAC inhibitors will be warranted.

This study is limited by the use of a single humanneuroblastoma cell line (SK-N-SH), which limits the gener-alizability of the results. The SK-N-SH line is non–n-mycamplified. It is well established that children without n-mycamplification have a better prognosis and are, therefore,presumed to have lower rates of clinical drug resistance. Forinstance, among children with relapsed neuroblastoma, thehazard ratio for death with n-myc amplification was 2.4 in amultivariable analysis of clinical and biologic factors associ-ated with overall survival [19]. Further work is, therefore,needed to determine if these results hold true in otherneuroblastoma cell lines, especially in 1 or more n-myc–amplified lines. In addition, although the triple combination ofdoxorubicin, vorinostat, and cambinol produced excessivetoxicity as quantified by animalweight and survival, we did notperform postmortem examinations to assess for organ-specifictoxicity. This information would be useful in future studies.Finally, further work is required to elucidate a potentialsynergistic effect between vorinostat and cambinol. Our studydesign did not include an in vivo group that received onlyvorinostat and cambinol, and triple therapy with these drugsplus doxorubicin produced toxicity, which precluded theassessment of this drug combination's efficacy.

Both the class III HDAC inhibitor cambinol and the classI/II HDAC inhibitor vorinostat demonstrated in vitro and invivo efficacy against doxorubicin-sensitive and DoxR neuro-blastoma. Although vorinostat has garnered much recentattention as a potential adjunct therapy for high-risk and

recurrent neuroblastoma and is currently under investigation ina number of clinical trials, cambinol appears to be at least aseffective and may be more potent against DoxR tumors.

References

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[2] Matthay KK, Reynolds CP, Seeger RC, et al. Long-term results forchildren with high-risk neuroblastoma treated on a randomized trial ofmyeloablative therapy followed by 13-cis-retinoic acid: a children'soncology group study. J Clin Oncol 2009;27:1007-13.

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[12] Heltweg B, Gatbonton T, Schuler AD, et al. Antitumor activity of asmall-molecule inhibitor of human silent information regulator 2enzymes. Cancer Res 2006;66:4368-77.

[13] Chu F, Chou PM, Zheng X, et al. Control of multidrug resistance genemdr1 and cancer resistance to chemotherapy by the longevity genesirt1. Cancer Res 2005;65:10183-7.

[14] Rebbaa A, Chou PM, Mirkin BL. Factors secreted by humanneuroblastoma mediated doxorubicin resistance by activating STAT3and inhibiting apoptosis. Mol Med 2001;7:393-400.

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