Dual Targeting of the Cyclin/Rb/E2F and Mitochondrial ... · some aspect of this pathway, for...

Cancer Therapy: Preclinical

Dual Targeting of the Cyclin/Rb/E2F and MitochondrialPathways in Mantle Cell Lymphoma with the TranslationInhibitor Silvestrol

Lapo Alinari1, Courtney J. Prince1, Ryan B. Edwards1, William H. Towns1, Rajeswaran Mani1,Amy Lehman2, Xiaoli Zhang2, David Jarjoura2, Li Pan3, A. Douglas Kinghorn3, Michael R. Grever1,Robert A. Baiocchi1, and David M. Lucas1,3

AbstractPurpose: During cell-cycle progression, D-cyclins activate cyclin-dependent kinases (CDKs) 4/6 to

inactivate Rb, permitting E2F1-mediated S-phase gene transcription. This critical pathway is typically

deregulated in cancer, and novel inhibitory strategies would be effective in a variety of tumors. The protein

synthesis inhibitor silvestrol has potent activity in B-cell leukemias via the mitochondrial pathway of

apoptosis, and also reduces cyclinD1 expression inbreast cancer and lymphoma cell lines.Wehypothesized

that this dual activity of silvestrolwouldmake it especially effective inmalignancies drivenby aberrant cyclin

D1 expression.

Experimental Design:Mantle cell lymphoma (MCL), characterized by elevated cyclin D1, was used as a

model to test this approach. The cyclinD/Rb/E2F1pathwaywas investigated in vitrousingMCL cell lines and

primary tumor cells. Silvestrol was also evaluated in vivo using an aggressive model of MCL.

Results: Silvestrol showed low nanomolar potency both inMCL cell lines and primaryMCL tumor cells.

D-cyclins were depleted with just 10 nmol/L silvestrol at 16 hours, with subsequent reductions of

phosphorylated Rb, E2F1 protein, and E2F1 target transcription. As showed in other leukemias, silvestrol

causedMcl-1 depletion followedbymitochondrial depolarization and caspase-dependent apoptosis, effects

not related to inhibition of CDK4/6. Silvestrol significantly (P < 0.0001) prolonged survival in a MCL

xenograft model without detectable toxicity.

Conclusions: These data indicate that silvestrol effectively targets the cyclin/CDK/Rb pathway, and

additionally induces cytotoxicity via intrinsic apoptosis. This dual activity may be an effective therapeutic

strategy in MCL and other malignancies. Clin Cancer Res; 18(17); 4600–11. �2012 AACR.

IntroductionInnormal cells, the progression fromG1- to S-phaseof the

cell cycle is tightly controlled by a conserved mechanisminvolving cyclins D1, D2, and/or D3, cyclin-dependentkinases (CDK) 4 and/or 6, CDK inhibitory proteins of theINK4 family, the tumor suppressor Rb, and transcriptionfactors of the E2F family. In nondividing cells, hypopho-

sphorylated Rb binds E2F proteins to suppress their activity.Upon appropriate signaling, D-cyclins bind and activateCDK4/6 to phosphorylate and inactivate Rb, freeing E2F toform a complex with other factors to drive the transcriptionof genes required for cell-cycle progression and DNA syn-thesis (reviewed in ref. 1). Nearly all tumors are defective insome aspect of this pathway, for example, through cyclinoverproduction, INK4 mutations, or Rb inactivation, pro-viding tumor cells a strong growth advantage and escapefrom normal mitotic control. Components of this pathwayare proposed to constitute valuable therapeutic targets (2,3), and considerable efforts are underway to develop spe-cific pharmacologic inhibitors. As an example, the CDK4/6-specific inhibitor PD-0332991 (4) has efficacy in a variety oftumor models (5–9), and is currently undergoing clinicaltesting (10, 11). However, as a single agent PD-0332991was reported to be cytostatic rather than cytotoxic, althoughit sensitizes cells to cytotoxic agents (6). Owing to the nearuniversal dysfunction of the cyclin/Rb pathway across can-cer types, a dual strategy to block the cyclin D/CDK4,6/Rbpathway, while concurrently activating apoptosis has thepotential to provide broad therapeutic benefit.

Authors' Affiliations: 1Department of Internal Medicine, College of Med-icine; 2Center for Biostatistics; and 3Division of Medicinal Chemistry andPharmacognosy, College of Pharmacy, The Ohio State University, Colum-bus, Ohio

R. A. Baiocchi and D. M. Lucas have made equal contributions to thisarticle.

Corresponding Authors: David M. Lucas, 410 West 12th Avenue,Room 455, Wiseman Hall, Columbus, OH 43210. Phone: 614-292-1551; Fax 614-292-3312; E-mail: [email protected] and RobertA. Baiocchi, 410 West 12th Avenue, Room 455, Wiseman Hall, Colum-bus, OH 43210. Phone: 614-292-1551; Fax 614-292-3312; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-12-0839

�2012 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 18(17) September 1, 20124600

on January 23, 2020. © 2012 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst July 12, 2012; DOI: 10.1158/1078-0432.CCR-12-0839

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A prime example of a tumor with a disrupted cyclin D/Rbaxis is the B-cell malignancy mantle cell lymphoma (MCL),in which the t (11; 14) (q13;q32) translocation placesCCND1, the gene for cyclin D1, under the control of animmunoglobulin promoter. This results in elevated andsustained cyclin D1 expression in tumor cells and concom-itant Rb inactivation, S-phase entry and cell division (12).In more aggressive cases mutations/deletions in the genesfor DNA damage response factors such as ataxia telangiec-tasia mutated (ATM) and p16ARF are likely to contribute toaberrant mitotic progression by impeding the activities ofCHK1/2 and p53 (13). MCL is a relatively uncommonsubset of Non-Hodgkin lymphoma, but accounts for adisproportionate number of deaths. Treatment options arelimited and relapses are nearly universal, highlighting theneed for new therapeutic approaches. Beyond the obviousclinical need, however, MCL provides an excellentmodel toinvestigate therapeutic targeting of the D-cyclin CDK4,6/Rbpathway.Silvestrol is a structurally unique, plant-derived cyclopen-

ta[b]benzofuran (14) with potent in vitro and in vivoantitumor activity in B-cell malignancies including acutelymphoblastic leukemia (ALL) and chronic lymphocyticleukemia (CLL) (15). Silvestrol is reported to block theinitiation step of translation by promoting an aberrantinteraction of the RNA helicase eIF4A with capped mRNA,thus preventing assembly into the eIF4F complex (16, 17).This effect leads to selective depletion of short half-lifeproteins, including Mcl-1 (15) and cyclin D1 (17, 18). Thetherapeutic benefit of protein synthesis inhibition in MCLand other B-cell malignancies is well-substantiated by thevast amount of data withmTOR inhibitors, and bothMcl-1and cyclin D1 are commonly shown to be affected by theseagents (19). Althoughmultiple studies show that inhibitingof either cyclin D1 alone (20) or CDK4/6 alone (5) is notcytotoxic, the resulting interference with tumor cell growth

in vivomaybe sufficient to provide therapeutic benefit.Moreimportantly, however, recent work indicates that inhibitionof theD-cyclin/CDK4,6 pathway can sensitize tumor cells totargeted agents including bortezomib (21) and imatinib(22). Thus, we hypothesized that silvestrol, through its dualactivities of D-cyclin inhibition and direct induction ofapoptosis, would be especially effective in rapidly prolifer-ating B-cell malignancies.

Here, we show that silvestrol shows potent cytostatic aswell as cytotoxic activity inMCL primary cells and cell lines.Low doses of silvestrol cause the loss of D-cyclins followedby Rb dephosphorylation and abrogation of E2F1-mediat-ed transcription. In addition, as we previously reported inchronic and acute lymphocytic leukemias, silvestrol inducesdepletion of Mcl-1 with subsequent mitochondrial depo-larization and apoptosis via the intrinsic pathway, thusproviding a dual antitumor effect. Importantly, silvestrolprovides a significant survival advantage in an aggressivemouse model of MCL. Together, these data support furtherpreclinical investigation of this novel agent in MCL as wellas other malignancies with a hyperactivated D-cyclin/CDK4,6 axis.

Materials and MethodsReagents

Isolation and characterization of silvestrol has beendescribed (14). The caspase inhibitor Q-VD-OPH (EnzymeSystems Products) was used at 20micromolar (mmol/L). JC-1 (5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazolyl-carbocyanine iodide) and dihydroethidium (DHE) wereobtained from Invitrogen. The CDK4/6 inhibitor PD-0332991 (4) was purchased from Active Biochem. TRAILwas purchased from R&D Systems.

Cells and cell linesMCL cell lines were kindly provided by Dr. Raymond Lai,

University of Alberta (Edmonton, AL, USA) and have beenpreviously described (23). PrimaryMCL cells were obtainedfrom the blood or marrow of patients diagnosed with MCLby World Health Organization criteria (24) after writteninformed consent according to the Declaration of Helsinki.Tumor cells were enriched to at least 85% by CD45 expres-sion (StemCell Technologies). All cells were incubated inRPMI 1640 supplementedwith heat-inactivated FBS (10%),L-glutamine (2 mmol/L), and penicillin (100 U/mL)/strep-tomycin (100 g/mL) (all from Sigma) at 37�C in a humid-ified atmosphere of 5% CO2.

Growth inhibition

To assess growth inhibition, CellTiter96� (MTS) assayswere carried out according to the manufacturer’s instruc-tions (Promega). IC50 values with 95% confidence intervalswere calculated using Prism (GraphPad Software).

Flow cytometry studiesCell viability was measured by flow cytometry using

annexin-V-FITC/propidium iodide (annexin/PI) according

Translational RelevanceTumor cells rely heavily on continued production of

proteins involved in growth, proliferation, and protec-tion from apoptosis. However, translation inhibitionrepresents anunderexplored approach in cancer therapy.Silvestrol is a unique agent that blocks translation direct-ly at the eIF4F complex, avoiding the compensatoryactivation of the Akt pathway as is seen with mTORinhibitors. Here, we show that silvestrol exhibits potentgrowth inhibitory activity inmantle cell lymphoma cellsand causes early depletion of cyclinD,with concomitantE2F1 deactivation and cell cycle arrest followed byapoptosis. These results potentially expand the impactof this work into diverse tumor types, most of which relyon sustained proliferation through defects in the cyclinD pathway. Thus, silvestrol may represent a proof-of-concept for an effective new therapeutic strategy incancer.

Targeting Translation in Mantle Cell Lymphoma

www.aacrjournals.org Clin Cancer Res; 18(17) September 1, 2012 4601




to the manufacturer’s instructions (BD Pharmingen). Mito-chondrial membrane depolarization was assessed usingJC-1, and reactive oxygen species (ROS) generation wasassessed using DHE as previously reported (25, 26). Cell-cycle studies were carried out according to standard proce-dures (27). All studies were conducted using a BeckmanCoulter FC500 instrument (Brea).

Immunoblot analysesSDS-PAGE and immunoblotting were carried out accord-

ing to standard procedures. Antibodies to cyclin D1 andMcl-1 were obtained from Santa Cruz Biotechnology;phosphorylated and total Rb from Cell Signaling Technol-ogy; E2F1 from Sigma; and GAPDH from Chemicon. Spe-cies-appropriate secondary antibodies conjugated withhorseradish peroxidase were obtained from Santa CruzBiotechnology. Protein bands were quantified by integra-tion of the chemiluminescence signals on an AlphaImagersystem (Proteinsimple).

Real-time reverse-transcription PCRTotal RNA was extracted using TRIzol (Invitrogen).

Real-time reverse-transcription PCR (RT-PCR) was car-ried out using TaqMan Universal Master Mix (AppliedBiosystems), using TATA binding protein (TBP) as anendogenous control. Primers and labeled probes wereobtained from Applied Biosystems. Mean threshold cycle(Ct) values were calculated by ABI PRISM software(Applied Biosystems) and used to determine fold differ-ences according to manufacturer’s instructions.

In vivo studiesThe JeKo-1 xenograft mouse model of MCL was used as

previously described by our group (28–30). In this model,mice develop an aggressive, widely disseminated lympho-ma with circulating and organ-infiltrating tumor cells and amedian survival of 4weeksposttumor injection.CB17SCIDmice (Taconic) were depleted of murine natural killer (NK)cells with weekly intraperitoneal injections of 0.2 mg anti-mouse interleukin-2 receptor b monoclonal antibody(TMb1), starting the day before engraftment. Mice wereinjected via the tail vein with 4 � 107 JeKo-1 cells, thenrandomized to 2 groups. Fifteen days postinjection, treat-mentwas initiated eitherwith vehicle (30%hydroxypropyl-b-cyclodextrin inwater) or silvestrol at 1.5mg/kg in vehicle,intraperitoneally every 48 hours. Animals were monitoreddaily for signs of tumor burden (weight loss, hind limbparalysis, respiratory distress, ruffled coat, and distendedabdomen). Animalswere euthanized if they exhibited eitherhind limb paralysis, 30% reduction in body mass, or 10%reduction in body mass together with respiratory distressand/or ruffled coat or lethargic behavior. The primaryendpoint was survival as defined by the lack of euthanasiacriteria. All animal work was reviewed and approved by theOSU University Laboratory Animal Resources (ULAR)-Institutional Animal Care and Use Committee. For thepharmacodynamics experiment, a subset ofmice at 3 weekspostengraftment were injected with either silvestrol or vehi-

cle. Spleen cells were obtained from euthanized mice at24 hours and lysates were immediately prepared. Immuno-blots for cyclin D1 were carried out as described earlier,using an identical aliquot of JeKo-1 lysate as a normalizingcontrol across blots.

StatisticsLinear mixed effects models were used to assess the

average interaction effect between TRAIL and all 5 silves-trol doses for Mino and Jeko-1 cells separately, using anadjusted a ¼ 0.025 level of significance for each compar-ison to control the overall type I error rate at a¼ 0.05. Forthe mouse experiments, Kaplan–Meier estimates of thesurvival function for vehicle and silvestrol conditionswere generated, and median survival times with 95%confidence intervals were calculated. Overall survivalbetween the 2 groups was compared using the log-ranktest using an a ¼ 0.05 level of significance. For thepharmacodynamics experiment, a mixed effects modelwas fit to account for experimental variability. SAS/STATsoftware (Version 9.2; SAS Institute Inc.) was used for allstatistical analyses.

ResultsSilvestrol has potent cytotoxic activity in MCL cells

The activity of silvestrol was examined using a panel ofMCL cell lines that included Mino, JeKo-1, and SP-53. Asshown in Fig. 1A, silvestrol showed low nanomolar IC50

values (concentration required for 50% growth inhibition)in the Mino and JeKo-1 cell lines at 48 hours by MTS assay.We previously reported that silvestrol is a substrate ofP-glycoprotein (31). As SP-53 cells were notably moreresistant to silvestrol, we hypothesized that P-gp expressionwas responsible for this difference. In support of this, SP-53 cells readily effluxed the fluorescent P-gp substrate rho-damine 123 (flow cytometry; data not shown), andthe 48hours IC50 was reduced approximately 4-fold in thepresence of the P-gp inhibitor verapamil (Fig. 1A). As theMTS assay does not distinguish cytotoxicity from cytostasis,cell death was confirmed in each case by annexin/propi-dium iodide (PI) staining and flow cytometric evaluation(Fig. 1B and additional data not shown). We next evaluatedthe effects of silvestrol in primary tumor cells from 6 MCLpatients (clinical characteristics of patients summarizedin Table 1). Cells were isolated by CD45 selection andincubated without or with 10 nmol/L silvestrol for 48hours, and cell viability was examined by annexin/PI flowcytometry (Fig. 1C). Data were plotted as percent live(annexin-negative and PI-negative) cells relative to eachsample’s time-matched untreated control. The results indi-cated variability in silvestrol sensitivity between patientsamples, but a median sensitivity comparable to theJeKo-1 and Mino cell lines. As we previously reportedthat silvestrol shows a relatively short half-life in vivo (32),we tested the effects with various exposure times. A 16hours exposure of JeKo-1 cells to silvestrol producedapproximately 50% of the effect achieved with continu-ous 48 hours exposure. Removing silvestrol after 24 hours

Alinari et al.

Clin Cancer Res; 18(17) September 1, 2012 Clinical Cancer Research4602




reduced its efficacy only minimally (Fig. 2; growth inhi-bition assessed at 48 hours in each case). Interestingly, at16 and 24 hours the cells remained largely viable (>90%and >80% annexin/PI negative, respectively; data notshown). These results are similar to our findings in CLLprimary cells (15), and together indicate that althoughthe cytotoxic effects of silvestrol require at least 8 to 12hours to initiate, they are largely irreversible by 24 hourseven though cell death as measured by PI is minimal atthat time. Our prior pharmacokinetics study (32) indi-cated that with a single-dosing strategy, concentrationsachievable in vivo for similar time periods remain in thelow nanomolar range. Thus, to reflect these data, furtherexperiments to assess the mechanism utilized 10 and 40nmol/L silvestrol for 16 and 24 hours.

Silvestrol depletes oncogenic proteins in MCL cellsCyclinsD1 andD3were dramatically reduced inMCL cell

lines with just 10 nmol/L silvestrol at 16 hours (Fig. 3A).Cyclin D2 could be detected in Mino, but not JeKo-1, cellsand appeared to be reduced with silvestrol treatment,although levels were insufficient to allow quantification(data not shown). Silvestrol also caused the loss of bothMcl-1 and c-myc, as was previously reported along withcyclin D1 in the Granta-519 MCL cell line and other tumortypes (15, 17, 18). Dramatic loss of cyclin D1 was alsoevident in primary MCL tumor cells with just 10 nmol/Lsilvestrol (Fig. 3B). These changes were not a consequenceof an apoptotic process; as mentioned earlier, viability ofcells at this time point (16 hours) were not different thanuntreated as determined by annexin/PI flow cytometry

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Figure 1. Efficacy of silvestrol in MCL cells. A, the indicated cell lines were incubated with various concentrations of silvestrol, and growth inhibition wasassessed at 48 hours by MTS assay (N � 3 each). In addition, the SP-53 cell line was retested under identical conditions in the presence of verapamil(10 mmol/L). IC50 and 95% confidence intervals are shown. B, JeKo-1 cells were incubated 24 hours as indicated, then analyzed by annexin (x-axis) andPI (y-axis) flow cytometry. Mino results were similar. Data are representative of 3 individual experiments. C, MCL patient samples (N ¼ 6; see Table 1)were incubated with silvestrol for 24 hours and evaluated by annexin/PI flow cytometry. Percent live (annexin and PI) are plotted relative to eachsample's untreated control. The horizontal black bar shows the median value.






(data not shown). However, depletion of each of theseproteins was not the result of transcriptional effects, ascyclin D1, Mcl-1 and c-myc mRNA levels were unchangedormoderately increased in these same samples (Fig. 3C). Ascyclin D1 is known to be degraded by the ubiquitin-protea-some pathway, we repeated this experiment in the presenceof the proteasome inhibitor bortezomib to determine if theloss of this protein was because of increased proteasomaldegradation. As shown in Fig. 3D, cyclin D1 depletion aftersilvestrol treatment was unaffected by the addition ofbortezomib. Together, these data are consistent with thehypothesis that silvestrol inhibits cyclin D1 production atthe translation stage.

Downstream effects of cyclin D1 depletionDuring cell-cycle progression, cyclin D1 translocates into

the nucleus and forms a holoenzyme with CDK4/6 tophosphorylate Rb, resulting in the release of E2F transcrip-tion factors and G1/S-phase transition (1). We thereforeassessed the effects of silvestrol-mediated cyclin D1 deple-tion in Mino and JeKo-1 MCL cell lines incubated for 16 or24 hours in the presence of 10 to 40 nmol/L silvestrol. AscyclinD1 depletion is expected to have the immediate effectof CDK4/6 inactivation, we also included the CDK4/6-specific inhibitor PD-0332991 (4) as a control. We carried

out preliminary experiments to determine the minimumeffective dose of PD-0332991 for these cell lines. Concen-trations between 100 and 1,000 nmol/L produced similareffects as measured by growth inhibition (MTS assay), andnone were cytotoxic as determined by annexin/PI flowcytometry (data not shown). We therefore used 100nmol/L in the remaining experiments. As expected, silves-trol and PD-0332991 both resulted in the loss of thephosphorylated form of Rb, although total Rb levels weregenerally not affected (Fig. 4A). In addition, silvestrolcaused a notable reduction in E2F1 protein (Fig. 4A). Thisreduction was also observed with PD-0332991, although toa lesser extent, suggesting that silvestrol-mediated E2F1 lossmight be partly attributable to loss of CDK4/6 activity aftercyclin depletion. Thus, silvestrol treatment may produceE2F1 inactivation both through hypophosphorylation ofRb as well as total E2F1 protein reduction. As shown in Fig.4B, message levels of classical E2F1 targets including cyclinE, proliferating cell nuclear antigen (PCNA), thymidinekinase, CDKs 2 and 4, MCM10, CDC45, and CDC6were notably reduced in Mino and JeKo-1 cells with just10 nmol/L silvestrol. Results using PD-0332991 were sim-ilar inMino cells, although in JeKo-1 cells PD-0332991 hadlittle effect on these targets at the 100 nmol/L concentrationused. These experiments also showed silvestrol-mediatedloss of E2F1mRNA. As E2F1 is regulated bymultiple factorsincluding cyclin D1 and c-myc as well as itself, this result isnot unexpected. Finally, low concentrations of silvestrolconsistently produced an increase in the G1 population anda decrease in the intermediate (S-phase) population, sug-gestive of arrest at the G1 transition (Fig. 4C).

Mechanism of cell deathAs loss of the Bcl-2 family member protein Mcl-1

is consistently observed in silvestrol-treated cells andE2F1 has been reported to modulate the transcriptionof apoptosis-related genes (33), we analyzed silvestrol-treated JeKo-1 and Mino cells for several of these factors.We did not observe substantial changes in protein levelsof Bcl-2, Bax, Bak, Bag-1, Bim, or XIAP (data not shown).Similar to what we previously reported in CLL and ALLcells, silvestrol treatment consistently induced mitochon-drial depolarization in JeKo-1 and Mino cells as early as16 hours that increased over time (24 hours datashown; Fig. 5A). This effect was not related to the

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Table 1. Characteristics of MCL patient samples

Pt no. Age/sex MCL variant StageSource oftumor cells

Previoustherapy

Symbolon chart

1 55/F Classic IVB lymph node No ^2 59/M Classic IVA blood No &3 65/M Classic IVB blood No ~4 52/M Blastoid IVB blood No x5 60/M Blastoid IVB blood Yes þ6 63/F Classic IVB blood Yes *

Alinari et al.





inhibition of CDK4/6, as 100 nmol/L PD-0332991produced little or no change. Addition of the caspaseinhibitor Q-VD-OPH reduced but did not prevent silves-trol-mediated mitochondrial depolarization, suggestingthe involvement of caspases in this process. Increases inROS production are indicative of mitochondrial pertur-bation. Therefore, we next evaluated silvestrol-inducedROS generation using Q-VD-OPH and gating around thelive population in the forward/side scatter to focus theanalysis on nonapoptotic cells. By 24 hours, ROS pro-duction was notably increased by silvestrol in both JeKo-

1 and Mino cells (Fig. 5B), indicating that silvestrolinduces ROS generation that is not simply a consequenceof cell death.

Loss of Mcl-1 is known to sensitize tumor cells to apo-ptosis mediated by TNF-receptor associated ligand (TRAIL;refs. 34 and 35). We therefore investigated whether silves-trol produced this effect. JeKo-1 and Mino cells were incu-bated 24 hours with or without 10 nmol/L silvestrol beforeadding TRAIL at various concentrations and incubating anadditional 24hours. In JeKo-1 cells, responseswere additivealthough a significant interaction effect (synergism)was not

Figure 3. Effects of silvestrol on keyMCL survival and proliferationproteins: A, JeKo-1 or Mino cellswere incubated 16 or 24 hours withsilvestrol as indicated, and lysateswere analyzed by immunoblot.Results are representative of at least3 separate experiments. Verticallines indicate deletion of an irrelevantlane in the same blot. B,MCL primarytumor cells were incubated 16 hours�silvestrol and immunoblotted forcyclin D1 (representative of 6 patientsamples).C,RNAwasextracted fromthe cells in (A) at the 16 hourstimepoint and analyzed by real-timeRT-PCR. Data were normalized toTBP and are shown relative to theuntreated time-matched sample.Results are averages of 3 separateexperiments, and the bars show�SD. D, JeKo-1 cells were incubatedwith or without silvestrol (10 nmol/L)or bortezomib (BTZ; 10 nmol/L) for 4hours (left 4 lanes). In addition, BTZwas added 2 hours before silvestroltreatment (right 2 lanes), andincubations continued for 4 hours.Mcl-1 was also included as a knownproteasome target. Lower bands inthe GAPDH blot are residual cyclinD1 (36 kDa). Similar results wereobserved in 2 additionalexperiments.

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observed (P ¼ 0.351; ref. Fig. 5C). However in Mino cells,the interaction effect across all doses was highly significant(P<0.0001), indicating amuch greater effect of TRAIL in thepresence of silvestrol (Fig. 5D). As TRAIL sensitization canalso be mediated by the loss of c-FLIP (36), we also inves-tigated c-FLIP expression by immunoblot. No changes inc-FLIP expression were observed in either cell line (data notshown).

In vivo activityWe and others previously showed that silvestrol pro-

duces significant in vivo benefit in diverse cancer modelsthat include prostate and breast cancer (17) and chronicand acute B-cell leukemias/lymphomas (15, 18). To eval-uate the single-agent activity of silvestrol in MCL, weemployed the JeKo-1/SCID model as recently reportedby our group (28). In this model, engraftment of JeKo-1cells produces an aggressive disseminated leukemia that,in untreated animals, results in a median survival of just28 days. For these studies, NK-cell–depleted SCID micewere injected intravenously with 40 million JeKo-1 cells.Day 15 postinoculation, treatments were initiated witheither vehicle alone (hydroxypropyl b-cyclodextrin, 30%in sterile water) or 1.5 mg/kg silvestrol in vehicle (n ¼ 10per group), administered intraperitoneally every 48hours. This dose was selected for its known safety instudies using nontumored animals (data not shown).The median survival of vehicle-treated versus silvestrol-treated mice was 27 versus 38 days (N ¼ 10 and 9respectively; log rank P < 0.0001; Fig. 6a). These results

were confirmed in a follow-up experiment using theidentical regimen, in which the median survivals were28.5 versus 36 days for vehicle- and silvestrol-treatedmice, respectively (N ¼ 10 each group; log rank P ¼0.0008). Importantly, there was no evidence of toxicity insilvestrol-treated animals as noted by weight loss; how-ever, detailed toxicity studies remain to be conducted.Separately, a cohort of identically engrafted animals wastreated 23 days postengraftment with a single injectioneither of silvestrol (N ¼ 10) or vehicle (n ¼ 9). Twenty-four hours later, spleen cells were collected for immuno-blotting. As shown in Fig. 6b, cyclin D1 levels weremoderately but significantly lower in the group of micereceiving silvestrol (0.47-fold versus vehicle; 95% CI ¼0.29–0.75; P ¼ 0.0112).

DiscussionMCL is an aggressive, incurable B-cell malignancy for

which novel therapeutic strategies are desperately needed.In addition, it represents a valuable model of disruptedcell-cycle control, a nearly universal characteristic of cancer.Thus, the use of MCL as a system to investigate mechanismsof tumor cell killing related to cell cycle may identify factorswith relevance to a variety of malignancies. Here, we showthat silvestrol potently induces cell growth inhibition andcell death in MCL cell lines and primary cells, and signif-icantly prolongs survival in an aggressive in vivo model ofMCL. These effects seem to be because of dual inhibition ofcell-cycle progression via depletion of D-cyclins and E2F1,as well as induction of mitochondrial depolarization and

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Figure 4. Effects of silvestrol oncyclin D downstream events. A,JeKo-1 and Mino cells wereincubated as indicated, and lysateswere analyzed for the indicatedproteins by immunoblot. TheCDK4/6 inhibitor PD-0332991(100 nmol/L) was included as acontrol. Results shown arerepresentative of at least 3experiments.

Alinari et al.





Figure 4. (Continued ) B, transcriptionaleffects of silvestrol on E2F1 targets inJeKo-1 (black bars) and Mino (grey bars)MCL cells as assessed by real-time RT-PCR. N ¼ 5 (except for cyclin E1, CDK2,and PCNA, N ¼ 4) and bars indicate�SD). C, cell-cycle effects of silvestrol inJeKo-1 and Mino MCL cell lines (16hours). All results are representative of atleast 3 individual experiments.

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intrinsic apoptosis via depletion of Mcl-1 as previouslyreported by our group (15). Although either cyclin Ddepletion (20) or CDK4/6 inhibition (6, 7) alone are notnecessarily sufficient to induce cell death, the data presentedhere suggest that the dual effect of cell-cycle inhibition andmitochondrial depolarization readily induces cell death,potentially representing an effective therapeutic strategy inMCL. Furthermore, this strategy sensitizes cells to addition-al agents such as TRAIL, as shows here, or to a variety ofchemotherapies (16, 18, 37).

Silvestrol seems to exert its effects via inhibition of trans-lation promoting an abnormal association of cappedmRNA with the RNA helicase eIF4A (16, 17), thus seques-

tering mRNA from the eIF4F initiation complex. This trans-lation inhibition mechanism likely explains the depletionof both cyclin D1 and Mcl-1 reported here, as mRNA forboth genes is unaffected or moderately increased and pro-teasome inhibition does not fully prevent protein loss.Agents with similar effects, but different mechanisms oftranslation inhibition, include sorafenib (38), homohar-ringtonine and its derivatives (39), and mTOR inhibitors(40). These agents have obvious therapeutic benefit inmultiple cancer types, and together with the data shownhere, strongly support translation inhibition as a valuabletherapeutic strategy. However, it will be important to ascer-tain the relative benefits of direct translation inhibitionwith

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Figure 5. Silvestrol mechanism ofcell death. A, JeKo-1 (top) andMino(bottom) cells were incubated 24hours as indicated and stainedusing the voltage-sensitive dyeJC-1. Mitochondrial integrity wasassessed as percentage of cellsremaining in the FL2 (y-axis) high,FL1 (x-axis) low population. Dataare representative of 3 separateexperiments. B, JeKo-1 and Minocells were incubated with orwithout silvestrol in the presence ofQ-VD-OPH (20 mmol/L). ROSproduction was analyzed by DHEflow cytometry. Resultsrepresentative of 3 separateexperiments.

Alinari et al.





silvestrol, versus indirect inhibition with various kinaseinhibitors. Hypothetically, direct inhibition of translationavoids the potential feedback activation of survival path-ways (41, 42), although we have not yet shown this toprovide an advantage in vivo. However, as both rapamycin(41, 42) and silvestrol (31) have been shown to activateresistance mechanisms, their optimal benefit is likely to beobserved in combination strategies.Silvestrol is a unique member of an intriguing class of

natural products, rocaglates, that are being investigated foractivity in hematologic malignancies alone or in combina-tion with other agents (37, 43, 44). Efficacy of this class ofagents in various solid tumors is also under evaluation, andour results showing inhibition of the D-cyclin/CDK4,6/Rbaxis suggest that it will have activity in a subset of these aswell. Interestingly, our results reveal subtle differencesbetween the effects of silvestrol in B-leukemias versus pros-tate or breast cancer cells, which might signal variations incytotoxic mechanisms despite the obvious potency of sil-vestrol in each of these tumor types. For example, as

reported by Cencic and colleagues (17), silvestrol causesloss of Bcl-2 protein expression inMDA-MB-231 breast andPC-3 prostate cancer cells, whereas our results show verylittle effect of silvestrol on Bcl-2 protein levels before celldeath, either in MCL as reported here, or in CLL or ALL(ref. 15 and data not shown). Bcl-2 is a critical antiapoptoticprotein and a validated therapeutic target, and its reductionin solid tumor cell lines suggests combination strategies ofsilvestrol with agents that show reduced efficacy in thepresence of elevated Bcl-2. However, the relative lack ofBcl-2 reduction in B-leukemias and lymphomas treatedwith silvestrol, coupled with their exquisite sensitivity tothis agent, indicates that Bcl-2 loss is not essential forsilvestrol-mediated cytotoxicity.

Previous reports suggest that rocaglates may act in part asinhibitors of theNF-kB pathway (45). AsNF-kB is frequent-ly activated in leukemias and serves a prosurvival function,

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Figure 6. Efficacy of silvestrol in vivo: SCID mice were NK cell-depletedwith weekly administration of anti-IL2R antibody, then engrafted with40� 106 JeKo-1 cells via the tail vein. A,micewere then randomized, and15 days postengraftment treatment was initiated with vehicle (N¼ 10) orsilvestrol at 1.5 mg/kg i.p. every 48 hours (N ¼ 9). Median survival wassignificantly prolonged (P < 0.0001). A repeat experiment showed similarresults (N ¼ 10 per group; P ¼ 0.0008). B, in a separate cohort, 3 weekspostengraftment mice were randomized to receive a single injection ofeither vehicle (N ¼ 9) or silvestrol (N ¼ 10). Twenty-four hours afterinjections, mice were euthanized and lysates prepared from spleen cells.Cyclin D1 levels were then assessed by immunoblot. Results show thecyclin D1 to actin ratios of each sample. Results were normalized acrossblots using an identical aliquot of JeKo-1 lysate.

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Figure 5. (Continued ) C, JeKo-1 and D, Mino cells were incubated24 hours without or with silvestrol (10 nmol/L). TRAIL was then added atthe indicated concentrations. After an additional 24 hours, cell growthinhibition was assessed by MTS assay. Data are shown relative to time-matched untreated cells. Data shown are the average of 3 separateexperiments, and bars show �SD. A significant synergistic interactionbetween silvestrol and TRAIL was observed in Mino (P < 0.0001) but notJeKo-1 (P ¼ 0.351) cells.






it would be valuable to find that silvestrol blocked thisimportant pathway. However, under the conditionsreported here, silvestrol treatment did not reduce mRNAlevels of classical NF-kB targets including XIAP, BCL2L1,NFKB1, CD74, and BCL3 (data not shown), and in factmoderate transcriptional increases were noted in putativeNF-kB targets CCND1, c-myc, and Mcl-1. This suggests thatNF-kB inhibition is unlikely to be a component of silves-trol-mediated cytostasis or cytotoxicity in MCL cells. Inaddition, although we observed the loss of the potentialtherapeutic target protein c-myc, classical c-myc-modu-lated genes were not found to be affected in these experi-ments. However, these observations do not rule out thepotential importance of silvestrol-mediated c-myc deple-tion in other tumor types.

The primary factors in the D-cyclin/CDK/E2F pathwayhave been reported to participate in cellular processes asdiverse as DNA damage response and repair, differenti-ation, and apoptosis, in addition to cell-cycle control(2, 3, 46). We have not yet evaluated the effect of silves-trol on each of these many pathways. Clearly, more

research is needed to sort out the consequences of inhi-biting D-cyclins and their downstream effector molecules.Regardless, our data show the strong therapeutic potentialof concurrently interfering with this plus other prosurvi-val pathways (i.e., mitochondrial stabilization) throughdirect inhibition of translation.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

AcknowledgmentsThe authors thank Yicheng Mao for excellent assistance with in vivo

experiments.

Grant SupportThis work was funded by the National Institutes of Health (NCI P50

CA140158). Silvestrol for all experiments was provided by A. D. Kinghorn(NCI P01 CA125066).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 12, 2012; revised May 21, 2012; accepted June 29, 2012;published OnlineFirst July 12, 2012.

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2012;18:4600-4611. Published OnlineFirst July 12, 2012.Clin Cancer Res Lapo Alinari, Courtney J. Prince, Ryan B. Edwards, et al. Mantle Cell Lymphoma with the Translation Inhibitor SilvestrolDual Targeting of the Cyclin/Rb/E2F and Mitochondrial Pathways in

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