Sensitizing B- and T- cell Lymphoma Cells to Paclitaxel ... · activities of AS101, demonstrating...

Cell Death and Survival

Sensitizing B- and T- cell Lymphoma Cells toPaclitaxel/Abraxane–Induced Death by AS101via Inhibition of the VLA-4–IL10–Survivin AxisHila Danoch1, Yona Kalechman1, Michael Albeck2, Dan L. Longo3, and Benjamin Sredni1

Abstract

Cancer cell resistance to chemotherapy is a major concern inclinical oncology, resulting in increased tumor growth anddecreased patient survival. Manipulation of apoptosis hasemerged as a new therapeutic strategy to eliminate cancer cells.The focus of this study resides within a novel approach to targetsurvivin, an integrator of both cell death andmitosis. This proteinplays a pivotal role in the resistance of tumors to chemotherapy,especially to paclitaxel. The data herein demonstrate an indirectrepressionof survivin inbothB- andT-cell lymphomaandhumanNHL by the nontoxic tellurium compound, AS101 [ammoniumtrichloro(dioxoethylene-o,o0)tellurate], via inhibition of tumorautocrine IL10–STAT3–Survivin signaling. As a result of survivinabrogation, sensitization of lymphomas to paclitaxel or to Abrax-ane, the new albumin-stabilized nanoparticle formulation of

paclitaxel, occurs both in vitro and in vivo. Importantly, inhibitionof lymphoma cell IL10 secretion is mediated by inactivation ofthe VLA-4 integrin, recently shown to be an important target ofAS101. This activity is followed by inhibition of the PI3K–AKTaxis that mediates IL10 suppression. Because a wide variety oflymphomas and other tumor types express VLA-4 and secreteIL10 in an autocrine manner, inhibition of survivin with a smallnontoxic agent has vast clinical significance in modulating che-mosensitivity in many tumor types.

Implications: Combination therapy with AS101 and paclitaxelhas novel therapeutic potential targeting deregulated activepathways in lymphoma, overcoming endogenous resistance toapoptosis. Mol Cancer Res; 13(3); 411–22. �2014 AACR.

IntroductionFrom the increasing evidence that dysregulation of apoptosis

contributes to in vivo drug resistance of tumor cells comes con-fidence that new therapeutic approaches aimed at correcting thedysfunctional apoptotic program could improve the treatmentoutcome of patients with chemotherapy-refractory tumors. Thepossibility of modulating the chemosensitivity of various cancercell types by targeting survivin has been actively pursued in the lastfew years. This protein is a structurally unique member of theinhibitor of apoptosis (IAP) family, which acts as a cell survivalfactor because it is involved in the control of mitotic progressionand inhibition of apoptosis (1) and is selectively expressedin most common human tumors (2). The selective overexpres-sion of survivin in cancer appears to reflect a global deregulation

of survivin gene transcription in transformed cells. This is consis-tentwith the cancer-specific transcriptionof the survivin promoter(3), and the several oncogenic pathways that converge to upre-gulate survivin gene expression in transformed cells. These includegrowth factor receptor signaling (4), Stat3 activation (5), PI3K/Aktsignaling (6), and loss of suppressor molecules such as p53 thatwas shown to transcriptionally repress survivin (7). Survivinexpression in normal tissues is developmentally regulated andthe protein was found to be absent or low in most terminallydifferentiated tissues (8). However, recent studies show thatalthough survivin is expressed in normal tissues characterizedby self-renewal and proliferation, its expression is significantlylower than in transformed cells (9, 10).

The differential expression in cancer and what appears to bea requirement to preserve tumor cell viability, identify survivinas a novel therapeutic target in cancer (11).

Growth autonomy of tumors appears as the result of multiplemechanisms by which growth regulatory pathways are deregu-lated. Malignant cells may produce excessive levels of autocrinegrowth factors, in a constitutive manner, which substitute forexogenous growth factor requirements. These factors may affectcritical genes that tightly regulate cell-cycle checkpoints, cellsurvival, and apoptosis. IL10 is spontaneously secreted by avariety of human cancer cells (12–16), including lymphoma(17). High IL10 levels have been detected in sera from patientswith a wide variety of solid and hematopoietic tumors(16, 18, 19). IL10 serum levels in patients with IL10-producingtumors were found to correlate with tumor progression and thepresence of distant metastases (19). Most importantly the cura-tive and noncurative surgery rates were correctly predicted by

1C.A.I.R. Institute, The Safdi�e AIDS and Immunology ResearchCenter, The Mina & Everard Goodman Faculty of Life Sciences,Bar-Ilan University, Ramat-Gan, Israel. 2Chemistry Department, Bar-Ilan University, Ramat-Gan, Israel. 3Laboratory of Molecular Biologyand Immunology, Biomedical Research Center, National Institute ofAging, Baltimore, Maryland.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

H. Danoch and Y. Kalechman contributed equally to this article.

Corresponding Author: Benjamin Sredni; Bar-Ilan University, Max&Anna Webb1, Ramat-Gan, 5290002, Israel. Phone: 972-3-531-8250; Fax: 972-3-738-4060;E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-14-0459

�2014 American Association for Cancer Research.

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preoperative IL10 serum levels in the majority of cases, withhigh cytokine serum levels paralleled by low rates of curativesurgery (14).

Stat3 is the main target of IL10 because constitutive Stat3activation upregulates survivin gene expression in malignant cells;it may be speculated that inhibition of IL10 in IL10-producingtumors, may result in sequential events leading to inhibition ofpStat3 followed by survivin repression.

The nontoxic immunomodulator ammonium trichloro(dioxo-ethylene-o,o0)tellurate (AS101) first developed by us, has beenshown to have beneficial effects in diverse preclinical and clinicalstudies. A large part of its activities has been primarily attributedto the indirect inhibition of the anti-inflammatory cytokineIL10 (20), followed by the simultaneous increase of specificcytokines. (21–23). Other tellurium derivatives developed by ushad no or low immunomodulating activity. These immunomod-ulatory properties were found to be crucial for the clinicalactivities of AS101, demonstrating the protective effects of AS101in parasite and viral-infected mice models (24), in autoimmunediseases (25), in septic mice (26), and in a variety of tumormodels in mice and humans where AS101 had a clear antitu-moral effect (21, 27). Phase I clinical trials on advanced cancerpatients treated with AS101 showed increased production andsecretion of a variety of cytokines, leading to a clear dominance inTh1 responses with a concurrent decrease in the Th2 responses(27). The predominance of Th1 responses was shown to berelated to AS101 antitumoral activity. All AS101 activities wereassociated with minimal toxicity. AS101 was shown to protectmice from hemopoietic damage caused by sublethal doses ofchemotherapy and increased the rate of survival of mice treatedwith different cytotoxic drugs acting by distinct mechanisms(22, 28, 29). In light of these results, phase II clinical trials withcancer patients treated with AS101 in combination with chemo-therapy have been initiated and completed, showing that treat-ment with AS101, with no major toxicity, induced a significantreduction in the severity of neutropenia and thrombocytopeniathat accompany chemotherapy (30). We have previously shownthat inhibition of IL10 by AS101 results in decreased Stat3activity in both solid tumor cells and rat glomerular mesangialcells (31, 32). This activity resulted in the sensitization of tu-mors to chemotherapy. Because of the central role of IL10inhibition in a large part of AS1010s preclinical activities, thepresent study offers a new approach to indirectly target survivinby a small tellurium molecule, which has demonstrated anexcellent safety profile in clinical trials in normal subjects andpatients with cancer. This approach is based on repression ofsurvivin by inhibition of the IL10–Stat3 cascade, resulting insensitization of tumors to chemotherapy. Because IL10, pStat3,and survivin are expressed in a wide variety of both hematologicand solid tumors, this study aims at examining the new approachof targeting survivin in both T and B lymphoma cells andelucidating the role of this activity in the sensitization of hema-tologic malignancies to Abraxane and paclitaxel. Abraxane is thenovel albumin-stabilized nanoparticle formulation of paclitaxel,designed to overcome insolubility problems encountered withpaclitaxel, and facilitating the passage of the drug to the under-lying tumor tissue. Albumin has the potential to increase drugdelivery to tumors by initiating albumin receptor (gp60)–medi-ated transcytosis across endothelial cells (33) and accumulatingdrug in tumors due to binding of albumin to secreted protein,acidic and rich in cysteine (SPARC).

Because resistance of many tumors to established treatmentregimens still constitutes a major problem in cancer therapy,novel strategies to target tumor cell resistance are essential toimprove patient outcome.

Elucidating the mechanism of survivin repression by AS101in hematologic malignant cells, and investigating the mechanismof tumor cell death ensuing from manipulation of the survivinpathway by AS101, may be relevant for novel therapeutic strat-egies aimed to improve the efficacy of chemotherapy-inducedapoptosis in patients.

Materials and MethodsReagents

All reagents are described in Supplementary Data.

AnimalsMale BALB/c 6 to 8 weeks of age, were purchased from Harlan

Laboratories. Animal experiments were performed in accordancewith approved institutional protocols and approved by the Insti-tutional Animal Care andUse Committee. A20 B lymphoma cellswere injected s.c. At day 10, when tumors were palpable, micewere treated as follows: controlmice treatedwith PBS on alternatedays. AS101 was injected on alternate days i.p. at 10 mg/mouse.Abraxane was injected at 0.5 mg/mouse on alternate days (�4).Survival of mice was monitored for 80 days.

Cell culturesThe mouse T (YAC) and B (A20) cell lymphoma cells and the

NHL cells were obtained from the American Type Culture Col-lection. Cells were cultured in RPMI-1640 containing 10% fetalcalf serum.

Attachment assayNinety-six-well plates were coated with 80-mL VCAM-1 (1

mg/mL). Cells with or without AS101 were incubated in thewells for 1 hour, Thereafter, cells were washed three times. Theattached cells were tested by the XTT at 450 nm.

Detection of apoptosisThe percentage of cells undergoing apoptosis was quantitative-

ly determined using an apoptosis detection kit (R&D Systems) byvirtue of their ability to bind Annexin V and exclude propidiumiodide (PI).

Evaluation of caspase activityDetermination of apoptosis was assessed by double staining

for Annexin V/PI using an apoptosis detection kit (Bender MedSystems Inc.). Activity of caspases was analyzed by FACS usingFLICA caspase activity kits (Alexis Biochemicals).

Cell-cycle distribution studiesCellswere treatedwith RNase and stainedwith PI.DNA content

was measured using a FACStar plus (Becton Dickinson) flowcytometer using Cell Quest software.

Quantitation of IL10Cells were cultured at various doses for different time points

with or without AS101. Supernatants were collected and evalu-ated by ELISA kits (R&D Systems).

Danoch et al.

Mol Cancer Res; 13(3) March 2015 Molecular Cancer Research412




Western blot analysisTotal cell extracts were prepared by suspension in ice-cold

lysis buffer containing 50 mmol/L Tris (pH 8), 150 mmol/LNaCl, 1%NP40, 0.1% SDS, 5mmol/L EDTA, 0.5% deoxycholate,0.2 mmol/L phenylmethylsulfonyl fluoride, 50 mmol/L NaF,200 mmol/L sodium vanadate, 5 mg/mL aprotinin, and 5 mg/mLleupeptin. Cell lysates were boiled and electrophoresed on10% SDS–PAGE and were then blotted with specific antibodies.Blots were developed using horseradish peroxidase–conjugatedsecondary antibodies and the ECL detection system (AmershamPharmacia Biotech).

Cell viability assayswere performed by the XTT test as describedin Ref. 34.

PlasmidspBabe-Akt (NM_005163) and the empty vector were a gift

from Dr. Ranan Berger (Sheba, Ramat-Gan, Israel), pECE-Stat3(GenBank U06922), and the empty vector were a gift fromProf H. Brody (Bar Ilan University, Ramat-Gan, Israel). PSM2cencoding IL10shRNA (NM_010548) and the empty vector(GE Healthcare). The tetracycline (or doxocycline) inducible(Tet-On) pSUPERIOR.retro.puro (OligoEngine) was used forsurvivin shRNA expression. These vectors are tetracycline-reg-ulated expression vectors that use regulatory elements from theEscherichia coli Tn10-encoded tetracycline (Tet) resistanceoperon. Tetracycline regulation in pSUPERIOR vectors is basedon the binding of tetracycline to the Tet repressor and dere-

pression of the promoter controlling expression of the gene ofinterest. The TetR-expressing vector pcDNA6/TR was usedfor regulation of gene transcription. ShSurvivin DNA inserts(NM_009689) were used. For transfection of vectors, poly-cationic transfection reagent (Lipofectamine;Invitrogen) wasused to facilitate uptake, according to the protocol recom-mended by the manufacturer.

Statistical analysisResults are expressed as mean � SE. For in vitro studies, differ-

ences between groups were analyzed using two-way ANOVA.Differences in survival curves between groups were calculated bythe Kaplan–Meier method. P < 0.05 was considered statisticallysignificant.

ResultsAS101 inhibits spontaneous IL10 secretion by certain B andT lymphoma cells

Figure 1 shows that both A20 (Fig. 1A; B-cell lymphoma) andYAC (Fig. 1B; T-cell lymphoma)murine cells constitutively secretehigh levels of IL10. The B lymphoma cells secreted higher levelsof IL10 (2,733 � 169 pg/mL) compared with T lymphoma cells(945�238pg/mL) following 48hours of incubation. Addition ofAS101 to cell cultures significantly decreased IL10 secretion byboth cell types in a dose-dependent manner amounting to 86.1%inhibition at 2.5 mg/mL AS101 in A20 cells (P < 0.01; Fig. 1A) and

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Figure 1.AS101 inhibits spontaneous IL10 secretion by A20 and YAC lymphoma cells. A20 (A) and YAC (B) lymphoma cells were cultured at 0.5 � 106/mL at 37�C inthe presence or absence of various AS101 concentrations. Supernatants were collected after 48 hours and tested by ELISA for IL10 content. Resultsrepresent mean � SE of three different experiments. � , P < 0.05 decrease versus control. �� , P < 0.01 decrease versus control. Accumulation of A20 (A) andYAC (B) cells in the sub-G1 fraction of the cell cycle following treatment with AS101 and paclitaxel. A20 and YAC lymphoma cells were cultured at 0.5 � 106/mLwith or without various concentrations of paclitaxel in the presence or absence of AS101 at 0.5 mg/mL (C and D) or with different concentrations ofAS101 in the presence of 30 nmol/L paclitaxel (E and F). Cell-cycle distribution was assessed after 24 hours of incubation and the accumulation ofcells in sub-G1 was determined. Results represent mean � SE of four different experiments. �, P < 0.05 and �� , P < 0.01 increase versus paclitaxel alone (C andD) and versus AS101 alone (E and F).

AS101 Sensitizes Lymphoma Cells to Paclitaxel/Abraxane

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to 74.5% in YAC cells (P < 0.01; Fig. 1B). The concentrations ofAS101 used did not affect cell viability (Fig. 1E and F). Similarresults of IL10 inhibition by AS101 were obtained at 24 hours ofincubation with control IL10 levels being lower than those at 48hours (data not shown).

AS101 sensitizes B- and T-cell lymphoma cells to paclitaxel-induced cell death

Figure 1C and D show that AS101 may increase the respon-siveness of cells from hematologic malignancies to the damagingeffect of paclitaxel. Figure 1 shows the response of both B (Fig. 1C)and T (Fig. 1D) lymphoma cells to paclitaxel in which a doseresponse accumulation of the cells in the sub-G1 fraction occurs.Although AS101 alone did not induce cell death, its coincubationwith paclitaxel (at 10–60 nmol/L) resulted in a significant shift

toward the sub-G1 fraction. The sensitization of B and T lympho-ma cells to paclitaxel-induced death and cycle alterations wasdependent on AS101 doses (Fig. 1E and F). In A20 cells (Fig. 1E),the dose of 2.5 mg/mL AS101 induced the highest paclitaxel-induced death. YAC cells were more sensitive to AS1010s effects.The low AS101 dose (0.5 mg/mL) affected maximally both para-meters (Fig. 1F).

The death induced by AS101 and paclitaxel in lymphoma cellshas apoptotic features

This was expressed by increased rate of three characteristics ofapoptotic cell death: annexin binding, PARP cleavage, and cas-pase-3 activity. Figure 2A shows negligible amounts of annexin-binding (non-PI-stained) YAC cells following treatment witheither AS101 or paclitaxel. However, cotreatment of YAC cells

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Figure 2.AS101 induces paclitaxel-induced apoptotic death. YAC cells were incubated in the presence of paclitaxel (30 nmol/L), AS101 (2.5 mg/mL), or the combinationof both for 24 hours. Apoptosis was assessed by AnnexinV binding (A) or by quantitation of caspase 3 activity (B and C) by FACS using FLICA caspaseactivity kits. A20 cells were incubated with various AS101 concentations with or without 30 nmol/L paclitaxel. Caspase-3 activity was assessed (D). PARP cleavagewas assessed in A20 cells subjected to AS101 and paclitaxel. The results of A, B, and E represent one of four independent experiments performed. The data inC and D represent mean � SE of four different experiments (E). #, P < 0.01 increase versus AS101 alone; � , P < 0.01 increase versus paclitaxel alone; �� , P < 0.001increase versus paclitaxel alone; ##, P < 0.001 increase versus AS101 alone.

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with AS101 and paclitaxel considerably elevated the proportionof annexin-positive, PI-negative cells. Figure 2B shows the sensi-tizing activity of AS101 to paclitaxel-induced apoptosis asreflected by increased caspase-3 activity in treated cells. Although0.14% and 1.23% of control and AS101-treated YAC cellsexpressed active caspase-3, 20.77% of paclitaxel-treated cellsexpressed this active cysteine protease. Nevertheless, coincuba-tionwith AS101 resulted in 86%of active caspase-expressing cells.This effect was dose-dependent in both T and B lymphoma cells(Fig. 2C andD). Analysis of A20 cells revealed that treatment withpaclitaxel considerably increased the expression of cleaved PARP.This increase was further amplified when cells were treated withAS101 and paclitaxel (Fig. 2E). These results collectively implythat AS101 sensitizes B and T lymphoma cells to paclitaxel-induced apoptotic death.

AS101 sensitizes B- and T-cell lymphoma cells to Abraxane-induced cell death

Abraxane, the new albumin-stabilized nanoparticle formu-lation of paclitaxel, was designed to overcome insolubilityproblems encountered with paclitaxel, facilitating the passageof the drug to the underlying tumor tissue. This drug appears torepresent an improvement in paclitaxel formulation in that itcan be administered safely without the risk of hypersensitivityreactions. Furthermore, the increased MTD and favorable tox-icity profile of Abraxane may ultimately prove advantageous interm of rate and quality of response. Thus, we tested thesensitizing effect of AS101 on Abraxane-induced lymphomacell death.

Supplementary Fig. S1 shows that AS101 sensitizes T (A) andB (B) lymphoma cells to Abraxane-induced death in a dose-dependent manner. Similarly to the results involving paclitaxel,presented in Fig. 2, YAC cells were more sensitive to thecombined treatment of AbraxaneþAS101, as compared withA20 cells. The combined treatment was effective starting with 5nmol/L Abraxane (Supplementary Fig. S1C). In these cells, theaccumulation of cells in the sub-G1 fraction increased inAbraxane dose-dependent manner (Supplementary Fig. S1). InA20 cells, the magnitude of the synergistic effect was lower.Treatment of YAC cells with AS101 combined with 60 nmol/LAbraxane resulted approximately 60% death when combinedwith AS101, compared with 9.2% death in Abraxane-treatedcells, while in A20 cells the maximal response amounted toapproximately 21% at the dose of Abraxane 70 nmol/LþAS101,compared with 6.3% in Abraxane-treated cells. Both types oflymphoma cells responded in an AS101 dose-dependent man-ner (Supplementary Fig. S1A and S1B).

AS101 sensitizes human breast carcinoma cells in vitro toAbraxane-induced death

Abraxane is now approved for the treatment of recurrentbreast cancer after combination chemotherapy or relapse with-in 6 months of adjuvant chemotherapy (33). We thereforewished to assess if AS101 may sensitize breast carcinoma cellsto Abraxane. For this purpose, we used human cultured MCF-7breast carcinoma cells, known to produce IL10. It can be clearlyseen that while AS101 does not affect cell viability, it signifi-cantly sensitizes cells to Abraxane in a dose-dependent manner(Supplementary Fig. S2A). Furthermore, AS101 significantlydownregulated IL10 secretion (Supplementary Fig. S2B), pstat3and srvivib expression (Supplementary Fig. S2C).

AS101 sensitizes T and B lymphoma cells to paclitaxel via IL10inhibition

Inhibition of IL10 has been previously shown to mediatemany of AS1010s activities. Here, we show that inhibition ofIL10 is indispensable for sensitizing the lymphoma cells stud-ied to paclitaxel. Figure 3A and B shows that treatment oflymphoma cells with paclitaxel combined with anti-IL10-neu-tralizing antibodies resulted in a similar sensitizing effect asAS101þpaclitaxel–induced death. Furthermore, this sensitiza-tion was abrogated in the presence of rIL10, suggesting thatsensitization of lymphoma cells to paclitaxel-induced deathby AS101 may be attributable to the inhibition of IL10. More-over, T leukemic cells that do not secrete IL10 (data not shown;T127 and Jurkat) were not sensitized by AS101 to paclitaxel-induced death (Supplementary Fig. S3A and S3B). To furtherascertain the role of IL10 inhibition in AS101-sensitizing effect,the IL10 gene was silenced by transfection with a plasmidexpressing IL10shRNA. Figure 3C shows the significant decreasein IL10 production by lymphoma cells transfected withthe silencing plasmid (from 630.67 � 30.18. to 158.9 �5.55 pg/mL; P < 0.001). AS101 significantly decreased IL10secretion by control but not by silenced cells (from 630.67� 30to 144 � 19.3 pg/mL; P < 0.001). Figure 3D shows that treat-ment of lymphoma cells transfected with the control plasmid,with either paclitaxel or Abraxane, resulted in a significantaccumulation of cells in the G2–M fraction. Addition of AS101combined with chemotherapy induced considerable apoptosisin treated cells. Nonetheless, treatment of IL10-silenced cellswith paclitaxel or Abraxane alone, induced extensive sub-G1

cell accumulation that was not further augmented followingaddition of AS101 (Fig. 3D). These results collectively suggestthat AS101 sensitizes lymphoma cells to Abraxane and pacli-taxel via inhibition of IL10 and probably not by additionaltargets.

Inhibition of the VLA-4 integrin activity on lymphoma cellsmediates IL10 inhibition by AS101

We have recently shown that AS101 inactivates the VLA-4integrin by redoxmodulation of vicinal thiols within the exofacialmembranal side (34). This activity accounted for the conversionof resistant to chemotherapy-sensitive acute myelogenous leuke-mia cells in vivo (34) and to favorable activities in murine modelsof autoimmune diseases (35, 36). Figure 4A shows that AS101inhibits the attachment of lymphoma cells to the VLA-4 ligandVCAM-1 in a dose-dependent manner, suggesting inhibition ofVLA-4 activity in these cells. Neutralization of VLA-4 by neutral-izing anti-VLA-4 antibodies inhibited IL10 secretion by lympho-ma cells (Fig. 4B) while combination with AS101 did not furtherdecrease this cytokine production, suggesting that AS101 andanti-VLA-4may act on a similar target. Inhibition of VLA-4 activityby AS101 was followed by dose-dependent decreased pAktexpression (Fig. 4C) and this effect was associated to the inhibi-tion of IL10 production by AS101. Figure 4D shows that the PI3Kinhibitor LY294002 inhibits IL10 secretion and this inhibition isnot further increased by addition of AS101, implying a commontarget for both compounds. To further substantiate this point, weshow that overexpression of Akt (Fig. 4E) abrogates the IL10inhibitory effect of AS101 on lymphoma cells (Fig. 4F). Theseresults collectively imply that regulation of IL10 in lymphomacells by AS101 ismediated by VLA-4 inactivation and the resultingpAkt decrease.






Mechanism of IL10-associated lymphoma cells sensitization byAS101Involvement of Stat3 in AS1010s activity. To gain further insightinto the mechanism of IL10-induced resistance to chemotherapyof lymphoma cells, and the consequences of its inhibition byAS101, we tested the role of Stat3 in these effects. The bestcharacterized IL10 signaling pathway is the Jak–Stat system. TheIL10–IL10R interaction engages the Jak family tyrosine kinasesJak1 and Tyk2 and induces tyrosine phosphorylation and activa-tion of the latent transcription factors Stat3, Stat1, and Stat5.However, Stat3 is used primarily by IL10 to carry out its signalinginto the cell. pStat3 is the main mediator of survivin upregula-tion. We first assessed the role of this phosphorylated protein inlymphoma cells resistance to chemotherapy and the consequenceof its potential inhibition by AS101 on their sensitization tochemotherapy by the compound. Figure 5A shows a dose-depen-dent decrease in the expression of phosphorylated Stat3, follow-ing addition of AS101 to lymphoma cells. The decrease in survivinexpression was more pronounced. Addition of rIL10 to cellcultures abolished the AS101-induced decrease in the expressionof both proteins, while treatment of cells with anti-IL10-neutral-izing antibodies had a similar effect to that of AS101. These resultscollectively suggest that AS101 downregulates the expression ofpStat3 and Survivin in lymphoma cells secreting IL10, and thiseffect is IL10-dependent.

The ability of AS101 to inhibit the expression of the abovementioned proteins led us to assess their role in the resistance oflymphoma cells to paclitaxel. We first used two known pStat3inhibitors: the JAK1 inhibitor piceatanol and the JAK2 inhibitorAG490. Figure 5B shows that both inhibitors are active at adose-dependant manner. As can be seen in Fig. 5C, treatment ofcells with either piceatanol or AG490 in combination withpaclitaxel significantly increased the sensitivity of lymphomacells to paclitaxel, similarly to cells treated with paclitaxelcombined with AS101. Collectively, these results suggest thatthe decrease in pStat3 by AS101 may account for its sensitizingeffect on these cells. Because we showed that inhibition oflymphoma cells IL10 by AS101 accounts for both its inhibitionof pStat3 and its sensitizing effects to paclitaxel, we assumedthat indeed AS101 sensitizes lymphoma cells to paclitaxel viainhibition of the IL10–pStat3 axis. To demonstrate this fact, weshowed that overexpression pf Stat3 in lymphoma cells signif-icantly abrogates the sensitizing effect of AS101 to paclitaxel-induced death (Fig. 5D). Accumulation of cells in the sub-G1

fraction decreased from 18.95% � 1.45% in cells transfectedwith the control vector and treated with AS101 (0.5 mg/mL)and paclitaxel to 7.6% � 0.75% in cells overexpressing Stat3(P < 0.05). At the higher dose of AS101 (2.5 mg/mL), thedecrease was more pronounced (from 42.11% � 0.49% to15.04% � 0.33%; P < 0.001).

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Figure 3.Inhibition of lymphoma IL10 mediates their sensitization by AS101 to paclitaxel-induced death. YAC cells (A) or A20 cells (B) were incubated for 24 hourswith 10 mg/mL aIL10 neutralizing antibodies, 30 nmol/L paclitaxel, 0.5 mg/mL AS101, or all three together, or with 50 ng/mL rIL10. The accumulationof cells in sub-G1 was determined; � , P < 0.01 increase versus paclitaxel; #, P < 0.01 increase versus paclitaxelþAS101; ^, P < 0.05 increase versuspaclitaxel. A20 cells were transfected with a plasmid encoding IL10shRNA or control scrambled shRNA (C). Cells were incubated with AS101, paclitaxel,Abraxane, or the combination of both for 24 hours. The accumulation of cells in sub-G1 was determined. Results represent mean � SE of four differentexperiments (D).

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AS101 sensitizes T and B lymphoma cells to paclitaxel viainhibition of survivin expression

The antiapoptotic protein Survivin has been previouslyshown to afford resistance to chemotherapy-induced death. Asseen in Fig. 5A, treatment of lymphoma cells with AS101 resultsin downregulation of pStat3 and Survivin. pStat3 is one ofthe main mediators of survivin upregulation. Because inhibi-tion of pStat3 by AS101 has been shown to mediate AS1010ssensitizing effect on lymphoma cells (Fig. 5D), we wishedto explore the role of survivin inhibition in AS1010s ability tosensitize lymphoma cells to chemotherapy. For this purpose,we used an inducible Tet-On expression vector system tocontrol the silencing of survivin by survivin–shRNA–expressingplasmid transfected into lymphoma cells. The inducible systemwas chosen because survivin is crucial for tumor cell survival,which precludes the option of permanently silencing the pro-tein in a stable transfection. Figure 6A shows that both thetransfection of survivin shRNA and the inducible system con-trolled by doxocycline were efficient in silencing survivinexpression. The scrambled shRNA served as negative control.AS101 significantly sensitized lymphoma cells transfectedwith scrambled shRNA, to paclitaxel-induced death, whethercells were treated with doxocycline (Fig. 6B) or not (Fig. 6C).Silencing survivin by survivin shRNA significantly sensitizedcells to paclitaxel (Fig. 6D), the percentage of viable cellsdecreasing from 82 � 2 (Fig. 6E) to 40 � 1 (Fig. 6D). Additionof AS101 to survivin-silenced cells did not further sensitize

them to paclitaxel-induced death (Fig. 6D), suggesting that thesensitizing effect of AS101 might be mediated by the sametarget protein as the silenced one, that is, survivin. Furthermore,the sensitizing effect of AS101 to Abraxane-induced lymphomacell death is probably also mediated by survivin inhibition. Ascan be seen in Fig. 7A, in lymphoma cells in which survivinwas not silenced (DOX�), Abraxane alone decreased the per-centage of viable cells by 20%, while its combined treatmentwith AS101 resulted in approximately a 60% decrease. In thepresence of doxocycline (DOXþ), Abraxane alone decreasedthe percentage of viable cells by 60% while addition of AS101did not further enhance cell death.

Collectively, the results show that AS101 sensitizes B and Tlymphoma cells to paclitaxel or Abraxane-induced apoptoticdeath by inhibition of the VLA-4–IL10–Stat3–survivin axis.

These in vitro results led us to evaluate whether AS101 maysensitize lymphoma cells to chemotherapy via inhibition of theIL10–Survivin axis also in vivo. For this purpose, mice wereinoculated with B lymphoma cells in which IL10 was silencedby stable transfection of IL10shRNA. Control cells were trans-fected with scrambled shRNA. As can be seen in Fig. 7B, only 20%of control mice survived 80 days after lymphoma cells inocula-tion. Although treatment with Abraxane increased survival rates,cotreatment with AS101 and Abraxane resulted in 90% survival. Itcan be clearly seen that silencing IL10 in B lymphoma cellssensitizes them to Abraxane in vivo (Fig. 7C). Eighty percent ofmice inoculated with IL10-silenced cells survived when treated

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Figure 4.Inhibition of lymphoma VLA-4 activity mediates IL10 inhibition by AS101. A20 cells were cultured with various AS101 concentrations on VCAM-1–coated platesfor 1 hour. The Percentage of attached cells was determined (A). A20 cells were cultured with 1 mg/mL AS101 or 1 mg/mL neutralizing anti-VLA-4 or controlantibodies, or the combination of AS101þaVLA for 48 hours. IL10 was quantitated in supernatants (B). A20 cells were incubated with AS101 for 24 hours,pAkt protein expression was quantitated (C). A20 cells were incubated with 1 mg/mL AS101, 50 mmol/L LY294002, or both combined for 48 hours. IL10 insupernatants was quantitated (D). A20 cells were transfected with either an empty vector or with a plasmid encoding Akt (pBabe-Akt; E). Each type of cellswas incubated with AS101 for 48 hours for quantitation of IL10 (F). C and E are one representative experiments of three performed. Results in A, B D, and F representmean � SE of three different experiments. � , P < 0.01 versus control; �� , P < 0.05 versus control.






with Abraxane alone, compared with only 50% of mice inocu-lated with control B lymphoma cells (Fig. 7B; P < 0.05). Further-more, no statistical difference was observed between the survivalof mice treated with Abraxane alone and that of mice treated withAS101 and Abraxane. These results strengthen the in vitro dataimplicating the IL10 axis as a major target of AS101 in sensitizingB lymphoma tumors secreting IL10, to chemotherapy-induceddeath. To provide clear relevance of the data to disease patho-genesis and therapy, we analyzed the mechanism of sensitizationin two human subtypes of NHL. Ramos and OCI-Ly19 cellsdiffered in the level of IL10 secreted. Diffuse large B-cell lympho-ma cells (OCI-Ly19) secreted significantly higher levels of IL10.Nevertheless, the secretion of IL10 by both subtypes of NHL wassignificantly inhibited by AS101 (Fig. 7D). The protein expressionof pStat3 and survivin also differed between the two NHL sub-types. The human diffuse large B-cell lymphoma cells expressedhigher levels of both proteins. As seen, AS101 clearly down-regulated the expression of both proteins (Fig. 7E). This wascorrelated with the cell response to paclitaxel. Although theOCI-Ly19 cells, expressing higher pStat3, surviving, and IL10,were resistant to 30 nmol/L paclitaxel, the Ramos cells responded

to chemotherapy, Nevertheless, AS101 significantly enhanced theresponsiveness of both cell subtypes to chemotherapy (Fig. 7F).

Coculture of both NHL cells subtypes with human BM stromalcells rendered them resistant to paclitaxel as opposed to thesignificant response of Ramos cells to chemotherapy. TreatmentwithAS101 sensitizes both the resistant and the responsive cells tochemotherapy in a dose-dependent manner. Importantly,because the survival of cells was performed on the mixture ofboth stromal and NHL cells together, Supplementary Fig. S4Bshows that stromal cells are not affected by the dose of paclitaxelused, implying the decrease in cell viability reflects that of NHLand not that of stromal cells. Furthermore, stromal cells were notsensitized to paclitaxel even in the presence of AS101 (Supple-mentary Fig. S4B). These data imply that AS101 may sensitizeNHL cells to paclitaxel at physiologic conditions.

DiscussionThis study proposes a novel approach to target survivin in B-

and T-cell lymphomas, a protein that plays a pivotal role in theresistance of tumors to chemotherapy, especially to paclitaxel.

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Figure 5.Role of pStat3 inhibition by AS101 in survivin repression by the compound. A20 cells were incubated with AS101, rIL10, or aIL10 for 24 hours. Cell extractswere evaluated for pStat3 and survivin protein expression by Western blot analysis (A). A20 cells were incubated with either AG490 or piceatanol for 24 hours.pStat3 protein expression was evaluated (B). A20 cells were incubated for 24 hours with 0.5 mg/mL AS101, 30 nmol/L paclitaxel, 50 mmol/L piceatanol,10 mmol/L AG490, or their combinations. Accumulation of cells in sub-G1 was evaluated (C). Transfected A20 cells overexpressing Stat3 (D) or control cellswere incubated with 0.5 or 2.5 mg/mL AS101, 30 nmol/L paclitaxel, or combinations for 24 hours. Accumulation of cells in sub-G1 was evaluated (D). Theresults in A and B represent one representative experiments of three performed. The data in C and D represent mean � SE of three different experiments.� , P < 0.01 versus paclitaxel; #, P < 0.01 versus AS101; �� , P < 0.01 versus control vector.

Danoch et al.





Our approach is based on the indirect repression of survivin bythe nontoxic tellurium compound, AS101, via inhibition oftumor autocrine IL10–Stat3–Survivin signaling. The inhibitionof this axis is mediated by inactivation of the VLA-4 integrin. Thestudy presents an attractive approach for anticancer therapy,having a vast significance in modulating chemosensitivity ofvarious tumor cell types not only lymphomas, for the reasonspresented below:

a. The differential expression of survivin in cancer.b. The wide variety of tumors, of both solid and hematologic

origin, that secrete IL10 in an autocrine manner, expressingconstitutively active Stat3.

c. The variety of tumors that highly express VLA-4.d. The use of a small molecule that has proved excellent safety

profile in humans.

In recent years, considerable efforts have been made tovalidate survivin as a new target in cancer therapy (37). Resultsfrom studies exploiting different strategies to interfere withsurvivin expression and function provided direct and convinc-

ing evidence that targeting the survivin network inhibitstumor growth potential in vitro and in vivo and increases sponta-neous and treatment-induced apoptosis of cancer cells, thusindicating survivin as a promising molecular target for cancertherapy (37).

Three independent approaches have been used for this aim inpreclinical testing:

a. Generation of an antigen-specific immune response againstsurvivin-bearing tumor cells for potential vaccination strate-gies (38).

b. Use ofmolecular antagonists, including antisense, ribozymes,siRNA, and dominant-negative survivin mutants results insuppression of tumor growth (39–41).

c. Mutation of Thr34 in survivin that abolishes a phosphoryla-tion site for the mitotic kinase p34cdc2, and results in adominant-negative phenotype, with induction of apoptosisand anticancer activity in vivo (40).

The present study introduces an alternative and novel modeto indirectly target survivin in IL10 secreting B and T lymphoma

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Figure 6.Role of targeting survivin by AS101 in the sensitization of lymphoma cells to paclitaxel-induced death. A20 cells were transfected with the tetracycline (ordoxocycline) inducible (Tet-On) pSUPERIOR.retro.puro vector expressing survivin shRNA or scrambled shRNA (A). Cells transfected with scrambled (B andC) or survivin (D and E) shRNA were incubated with AS101, paclitaxel, or the combination of both for 24 hours and were either induced (B and D) or notinduced (C and E) by doxocycline. Viability of the cells was assessed by the XTT assay. The data represent mean � SE of three different experiments.� , P < 0.05 decrease versus control; �� , P < 0.01 decrease versus control; ##, P < 0.01 decrease versus paclitaxel.






cells by a nontoxic tellurium compound currently used inclinical studies. This is the first time in which the VLA-4–IL10axis is proven to regulate survivin expression in malignant cells.

Studies have shown that under normal circumstances, cells usedifferent death mechanisms to maintain homeostasis and phys-iologic integrity of the organism, but these processes often appearto be altered in cancer. Thus, in recent years, developing variousstrategies for administration of cytotoxic chemotherapeutics incombination with apoptosis-sensitizing reagents is receivingmore emphasis (41).

The study provides sound evidence on the sequential eventsoccurring following treatment of lymphoma cells with AS101.These events initiate by interaction of AS101 with the VLA-4integrin and terminate with the inhibition of survivin expressionby lymphoma cells resulting in increased sensitivity of lymphomacells topaclitaxel or Abraxane.Wehave recently shown thatAS101inactivates the VLA-4 integrin by redox modulation of vicinalthiols within the exofacial membranal side (34). This activityaccounted for the conversion of resistant to chemotherapy-sen-sitive acute myelogenous leukemia cells in vivo (34) and to

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Figure 7.Role of targeting survivin by AS101 in the sensitization of lymphoma cells to Abraxane-induced death and the role of IL10 inhibition in sensitization oflymphomas to Abraxane in vivo. A20 cells were transfected with the tetracycline (or doxocycline) inducible (Tet-On) pSUPERIOR.retro.puro vectorexpressing survivin shRNA and incubated as described in A. Cells were either induced or not induced by Doxocycline. Cell viability was assessed after24 hours by the XTT assay. � , decrease versus control DOXþ; $, P < 0.05 versus control DOX�; #, P < 0.05 decrease versus Abraxane DOX�. Thedata represent mean � SE of three different experiments (A). A20 cells expressing control (B) or IL10shRNA (C) were implanted into mice. At day10, when tumors were palpable, mice were treated as follows: control mice treated with PBS on alternate days. AS101 was injected on alternate days i.p. at10 mg/mouse. Abraxane was injected at 0.5 mg/mouse on alternate days (�4). Survival of mice was monitored for 80 days. The data represent atotal of 10 mice/group. @, P < 0.05 increase versus PBS or AS101 or Abraxane; #, P < 0.05 increase versus PBS or AS101; �, P < 0.05 increase versus PBS.Ramos and OCI-Ly19 NHL cells were cultured at 0.5 � 106/mL at 37�C in the presence or absence of various AS101 concentrations. Supernatants werecollected after 48 hours and tested by ELISA for IL10 content. Results represent mean � SE of three different experiments. � , P < 0.05 decreaseversus control; �� , P < 0.01 decrease versus Ramos cells. D, Ramos and OCI-Ly19 NHL cells incubated with AS101 for 24 hours. pStat3 and survivinprotein expression was quantitated (E). Ramos and OCI-Ly19 NHL cells were cultured at 0.5 � 106/mL with or without 30 nmol/L paclitaxel in the presenceor absence of AS101 (F). Cell viability was assessed after 24 hours of incubation by the XTT assay. Results represent mean � SE of four different experiments.� , P < 0.05 versus "without paclitaxel" and �� , P < 0.01 versus control.

Danoch et al.





favorable activities in murine models of autoimmune diseases(35, 36). Indeed, we show here that the compound inhibits theactivity of VLA-4 on lymphoma cells as expressed by inhibition ofcell attachment to the VLA-4 ligand VCAM-1. The VLA-4 integrinhas been recently found to be expressed on lymphoma cells (42).We show in this study that inactivation of VLA-4 mediates theinhibitory effect of AS101 on lymphoma IL10 production Spe-cifically, our data imply that regulation of IL10 in lymphoma cellsby AS101 is mediated by VLA-4 inactivation and the resultingdecrease in pAkt. The essential role of PI3K–Akt in IL10 produc-tionmay be explained by deactivation of GSK3b (43). GSK3b hasbeen reported to decrease the nuclear translocation of transcriptorfactors that are crucial for IL10 expression including CREB, C/EBPb, C/EBPV, and NF-kB (44)

The study focuses on the mechanism of survivin repression byAS101 and its impact on paclitaxel or Abraxane-induced lym-phoma cell death. Our drug choice consisted on the fact thatpaclitaxel is one of the most effective cancer chemotherapeuticagents to date. The dual role of survivin in apoptosis inhibitionand in regulation of spindle dynamics and microtubule integritymay facilitate evasion from checkpoint mechanisms of cell-cyclearrest and promote resistance to microtubules interfering chemo-therapeutic drugs such as paclitaxel (45).

Part of our data is in line with other studies. Numerous studieshave detected constitutively active Stat3 in diverse human tumorspecimens and established persistent Stat3 activity as essential formalignant transformation of cultured cells by many oncogenicsignaling pathways (46). Activated Stat3 has been previouslyshown to regulate survivin expression (47) Inhibition of IL10 byAS101 has been previously shown to sensitize tumors to chemo-therapy (33). Nevertheless, this is the first report in which thesequential events starting by VLA-4 inactivation resulting in IL10repression, inducingdecreased pStat3 expression ensuing survivinrepression, are demonstrated to be interdependent in the sensi-tization of mice and human lymphoma cells to paclitaxel orAbraxane. Other targets of Stat3 and Akt, such as BCl2, P21waf,or p53, might be theoretically also modulated.

Besides our prototype tellurium compound AS101, the inves-tigation of therapeutic activities of other tellurium(IV) com-pounds is scarce in the literature, although tellurium is the fourthmost abundant trace element in the human body. Our integratedresults show sensitization of IL10-secreting lymphoma cells topaclitaxel or Abraxane-induced death can be achieved by a uniquealternative approach to existing strategies of controlling survivin,a pivotal molecule at the junction of cancer cell survival and

division networks. This regulation may be achieved using AS101,currently being tested in clinical trials and might be beneficial inthe treatment of patients with lymphoma with high functionalVLA-4 expression and high level of IL10.

Unlike most conventional chemotherapies, taxanes, such asdocetaxel and Abraxane, have been shown to upregulate survivinexpression levels, which is attributed tomitotic arrest at the G2–Mstage and the augmented stability of survivin protein (48). Inclinical settings, docetaxel and paclitaxel treatment is effectiveagainst several types of human cancers. Despite their clinicalbenefits, the long-term treatment of these drugs is often limitedbecause of development of drug resistance and cumulative sideeffects, primarily neuropathy and myeloid toxicity. Therefore,drugs that circumvent paclitaxel or docetaxel resistance withoutoverlapping side effects represent ideal candidates for developingnovel combinatorial therapeutic regimen for lymphomas. Ourstudy suggests that survivin is an important factor that counteractspaclitaxel/Abraxanel–induced apoptosis in lymphoma cells andthis protein can be distinctively counteracted by AS101.

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

Authors' ContributionsConception and design: H. Danoch, Y. Kalechman, M. Albeck, D.L. Longo,B. SredniDevelopment of methodology: H. Danoch, M. AlbeckAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H. Danoch, D.L. Longo, B. SredniWriting, review, and/or revision of themanuscript: Y. Kalechman,D.L. Longo,B. SredniAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H. DanochStudy supervision: B. Sredni

Grant SupportThis work was partly supported by The Safdi�e Institute for AIDS and

Immunology Research, The Milton and Lois Shiffman Global Research Pro-gram, The Dave and Florence Muskovitz Chair in Cancer Research, The CometWalerstein Cancer Research Program and the DorshaWallman Cancer ResearchEndowment, and Frida Stollman Cancer Memorial Fund.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 18, 2014; revised October 6, 2014; accepted October 15,2014; published OnlineFirst October 28, 2014.

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Danoch et al.




2015;13:411-422. Published OnlineFirst October 28, 2014.Mol Cancer Res Hila Danoch, Yona Kalechman, Michael Albeck, et al. Axis

Survivin−IL10−Induced Death by AS101 via Inhibition of the VLA-4−Sensitizing B- and T- cell Lymphoma Cells to Paclitaxel/Abraxane

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