Cap-Translation Inhibitor, 4EGI-1, Restores Sensitivity toABT-737 Apoptosis through Cap-Dependent and-Independent Mechanisms in Chronic LymphocyticLeukemia
Shaun Willimott1, Daniel Beck1, Matthew J. Ahearne1,2, Victoria C. Adams1,2, and Simon D. Wagner1
AbstractPurpose: The lymph node microenvironment promotes resistance to chemotherapy in chronic
lymphocytic leukemia (CLL), partly through induction of BCL2 family prosurvival proteins. Currently
available inhibitors do not target all BCL2 family prosurvival proteins and their effectiveness is also
modified by proapoptotic BCL2 homology domain 3 (BH3) only protein expression. The goal of this
study was to evaluate synergy between the eIF4E/eIF4G interaction inhibitor, 4EGI-1, and the BH3
mimetic, ABT-737.
Experimental Design: CLL cells were cultured in conditions to mimic the lymph node microenviron-
ment. Protein synthesis and cap-complex formation were determined. Polysome association of mRNAs
fromBCL2 family survival geneswas analyzedby translational profiling. The effects of 4EGI-1 and theBCL2/
BCL2L1 antagonist, ABT-737, on CLL cell apoptosis were determined.
Results: Protein synthesis was increased approximately 6-fold by stromal cell/CD154 culture in a
phosphoinositide 3-kinase a (PI3Ka)–specific manner and was reduced by 4EGI-1. PI3K inhibitors and
4EGI-1 also reduced cap-complex formation but only 4EGI-1 consistently reduced BCL2L1 and BCL2A1
protein levels. 4EGI-1, butnotPI3K inhibitors or rapamycin, inducedanendoplasmic reticulumstress response
including proapoptotic NOXA and the translation inhibitor phosphorylated eIF2a. 4EGI-1 and ABT-737
synergized to cause apoptosis, independent of levels of prosurvival protein expression in individual patients.
Conclusions: Overall protein synthesis and cap-complex formation are induced by microenvironment
stimuli in CLL. Inhibition of the cap-complex was not sufficient to repress BCL2 family prosurvival
expression, but 4EGI-1 inhibited BCL2A1 and BCL2L1 while inducing NOXA through cap-dependent and
-independent mechanisms. 4EGI-1 and ABT-737 synergized to produce apoptosis, and these agents may be
the basis for a therapeutically useful combination. Clin Cancer Res; 19(12); 3212–23. �2013 AACR.
IntroductionDefects in apoptosis due to dysregulation of BCL2 family
proteins are common in cancers and BCL2 homologydomain 3 (BH3) mimetic drugs, such as ABT-263, whichbind and inhibit antiapoptotic BCL2, and to a lesser extent
BCL2L1, are being trialed in lymphoid malignancies (1, 2).ABT-263 was derived from a prior compound, ABT-737,and several causes of resistance to ABT-737 that may limitthe clinical use of this class of agents are recognized. Forexample, increased expression of antiapoptotic proteinsBCL2L1 (formerly BCL-XL; refs. 3, 4), BCL2A1 (formerlyA1 or BFL1; refs. 4, 5), and MCL1 (5–7) reduce the effec-tiveness of ABT-737 and extracellular signal–regulatedkinase (ERK) signaling pathways also promote resistanceto this agent (8).
Circulating chronic lymphocytic leukemia (CLL) cellsexpressmore BCL2 thannormal B cells and in vitro leukemiccells are highly sensitive to ABT-737 (4, 7), but withinlymph nodes leukemic cells express BCL2A1, BCL2L1(9), andMCL1 (10),which promote resistance to this agent.Induction of these proteins is likely to be due to signalsfrom the lymph node microenvironment (11) and in sup-port of this, CLL cells stimulated in vitro by the T-cell surfacemolecule, CD154 (9, 12) or through B-cell receptor cross-linking (13) reproduce these expression changes. CLL is
Authors' Affiliations: 1Department of Cancer Studies and MolecularMedicine and MRC Toxicology Unit, University of Leicester; and 2Depart-ment of Haematology, University Hospitals of Leicester, Leicester, UnitedKingdom
Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).
S. Willimott and D. Beck contributed equally to this work.
CorrespondingAuthor:SimonD.Wagner,MRCToxicologyUnit, HodgkinBuilding, University of Leicester, Room323, Lancaster Road, Leicester LE19HN, United Kingdom. Phone: 44-1162525584; Fax: 44-1162525616;E-mail: [email protected]
incurable with conventional chemotherapy and robustsurvival within the lymph node microenvironment is con-sidered to be a cause of treatment failure. Consequently,there is interest in evaluating agents targeting microenvi-ronment-induced signals for treatment of CLL (11).Translation is increased in transformed cells and cancers
(14) and several studies have shown that overexpressionof one component of the cap-binding complex, eIF4E, issufficient to cause cellular transformation (15–17) andtransformation of primary embryonic fibroblasts (18) inpart by cooperation with c-MYC. Specifically in B cells,overexpression of the cap-binding complex promotes lym-phomagenesis, again in cooperation with c-MYC, in eIF4Etransgenic mice (19, 20). eIF4E levels are elevated in non-Hodgkin’s lymphoma and are also high in normal germi-nal centers with little expression in the mantle zone (21).Most cases of clinically aggressive lymphoma show strongexpression of eIF4E (21). Therefore, increased expressionof components of the translation machinery is sufficientto support lymphomas in experimental systems. Proteintranslation is a target for treatment in cancer (22) andhematologic malignancies (23), and the eIF4E inhibitorand antiviral agent ribavirin has been trialed in acutemyeloid leukemia (24).There are indications that inhibition of translation may
be a useful strategy to induce apoptosis in CLL. Two naturalproducts–homoharringtonine (25) and silvestrol (26)–thatexert their effects partly through inhibition of translationcause apoptosis of CLL cells in vitro and in vivo. Homo-harringtonine prevented the prosurvival effects of stromalcell culture. However, both these compounds have effects
on many aspects of cellular metabolism apart fromtranslation.
We identify increased protein synthesis as being animportant feature of CLL cells supported on stromalcells/CD154. We also show that a specific inhibitor ofeIF4E/eIF4G interaction, 4EGI-1, causes changes to BCL2family proteins favoring synergy with a BCL2/BCL2L1antagonist, ABT-737, but part of this effect is independentof cap-dependent translation through induction of anendoplasmic reticulum (ER) stress response. We suggestthat this combination of agents will be a useful route toabrogating the prosurvival effects of themicroenvironment.
Materials and MethodsPatient samples
CLL cells were isolated from whole blood using densitygradient centrifugation. Patients included those with bothearly stage and advanced disease. Only patients with whitecell count more than 50 � 109/L were used in the study. Atthe time of study, no patient had been treated for 3months.Local research ethics committee approval was obtained.Patient characteristics are presented in Table 1.
Cell cultureCLL cells (3 � 106/mL) were cultured in RPMI-1640
medium (Invitrogen) supplemented with FBS (Invitrogen),nonessential amino acids (Invitrogen), penicillin/strepto-mycin (Invitrogen), and HEPES buffer (Lonza) in a 37�Cand 5% CO2 incubator. The cells were either cultured ontissue culture plastic or cocultured with 80% to 90% con-fluent and35Gy irradiatednontransfectedmousefibroblastcells (NT culture) or human CD154-expressing mousefibroblast cells supplemented with rh-IL-4 (10 ng/mL; R&DSystems; CD culture; ref. 27).
The phosphoinositide 3-kinase (PI3K) inhibitors,LY294002 (Calbiochem), PI-103 (Calbiochem), and PIK-294 (Calbiochem) andBH3-mimetic, ABT-737were used asindicated. Rapamycin was purchased from Sigma. 4EGI-1(28) and salubrinal (29) from Santa Cruz Biotechnology.
Sucrose density gradientsCycloheximide (100 mg/mL; Sigma) was added before
harvesting 36 to 40� 106 cells. Following lysis in 500 mL ofpolysome extraction buffer [15 mmol/L Tris (pH 7.5),15 mmol/L MgCl2, 300 mmol/L NaCl, 1% Triton X-100,100 mg/mL cycloheximide, 50 mg/mL heparin, 5 mmol/Ldithiothreitol, and RNase inhibitors] lysates were centri-fuged and the supernatant layered on to a 10% to 50%sucrose gradient [Biocomp Gradient Station (Wolf Labo-ratories Ltd.)], followed by centrifugation at 38,000 rpmfor 2 hours in a Beckman SW40 rotor. mRNAwas extractedfrom each of 12 fractions using phenol/chloroform ex-traction and lithium chloride precipitation. TaqMan real-time PCR assays were from Applied Biosystems [actin(ACTB) #Hs99999903_m1, RPS6 #Hs04195024_g1 MCL1#Hs03043898_m1, BCL2A1 #Hs00187845_m1, BCL2L2#Hs01573809_g1]. The relative amount of mRNA in
Translational RelevanceThe tissue microenvironment contributes to surviv-
al, proliferation, and resistance to conventional che-motherapy in chronic lymphocytic leukemia (CLL),and abrogating the effects of the microenvironment isa goal of therapy. There is interest in using small-molecule BCL2/BCL2L1 antagonists for the treatmentof cancers, but such agents do not inhibit prosurvival,MCL1 and their effectiveness is also critically depen-dent on BCL2 homology domain 3 (BH3)–only proa-poptotic protein expression. We showed that a culturesystem mimicking the microenvironment increasedprotein synthesis and this prompted us to evaluate4EGI-1, a small-molecule inhibitor of cap-complexformation. This agent repressed prosurvival BCL2L1and BCL2A1 and induced proapoptotic NOXA, a pat-tern of activity suggesting useful cooperation with theBH3 mimetic, ABT-737. We showed synergy between4EGI-1 and a BCL2/BCL2L1 antagonist in differentindividuals across a range of expression levels. Com-bination of 4EGI-1 with BCL2/BCL2L1 antagonistsmay be highly effective in targeting leukemic cellswithin the microenvironment.
Cap-Translation Inhibition in CLL
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Patient Age Gender Stage CD38 U/M VH gene Cytogenetics
1 45 F A 1 ND ND ND
2 69 F A ND M 3-07 ND
3 61 M A ND M 4-b
4 77 M A 1 M 4-31
5 61 M A 7 ND ND
6 87 M A 1 ND ND
7 69 M A 1 M 3-07 13q�8 59 M A 1 M 3-23 13q�9 83 F C 50 M 3-21 13q�, 11q�10 79 M A 100 M 3-23 13q�11 76 F A 8 M 4-34 13q�12 82 M A 100 M 3-48 13q�13 68 M B ND M 4-34 13q�14 65 F A 17 U 1-69 13q�15 87 F B ND M 4-59 ND
16 77 F A ND U 4-61
17 73 M A 3 U 4-31 13q�, 11q�18 69 M A 30 U 3-33 del 6q and
add(6)(q13–14)
19 57 M B 16 U 1-69 13q�20 68 M A ND ND ND ND
21 50 M ND ND U 1-69 t12
22 43 M B ND M 3-21
23 60 M A 2 M 3-07 13q�, 17p�24 69 M A ND U 4-39
25 63 F A ND ND ND
26 80 F A ND M 3-23 13q�27 67 M A ND M 4-34
28 84 F A ND ND ND
29 90 F A 36 ND ND ND
30 74 F A 10 M 4-34 13q�31 57 M A ND M 3-15
32 62 F A 83 M 3-23
33 81 M B 100 U 1-69 13q�, 11q�34 52 M B 16 U 1-69 13q�35 56 M A ND M 1-18
36 62 M A 1 U 3-23
37 71 F A 10 M 3-07
38 73 M A 32 U 3-30
39 69 F A 1 M 2-05 13q�40 61 M C 2 M 4-28
NOTE: Patient gender, clinical stage according to the Binet classification, percentage of cells expressing the CD38 surface marker,immunoglobulin genemutational status [either unmutated (U) ormutated (M)], VH gene segment and cytogenetics, whichwas obtainedeither by using a FISH panel or by analysis of metaphase spreads. Blanks under cytogenetics means no abnormalities detected byFISH panel.Abbreviation: ND, not determined.
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each fraction of the sucrose gradient was expressed as afraction of the total amount of mRNA on the gradient.
Cap-binding assayCLL cells (50 � 106) were cultured for 24 hours on
CD154/IL-4 in the presence or absence of LY294002 (20mmol/L). Cells were harvested, washed in 300 mL ice-coldbuffer A [50 mmol/L MOPS/KOH, pH 7.2, 50 mmol/LNaCl, 50 mmol/L NaF, 2 mmol/L EGTA, 5 mmol/L EDTA,7 mmol/L 2-mercaptoethanol, and protease and phospha-tase inhibitors (Sigma)], and lysed by the addition of 1.5%(v/v) NP-40 and 1.5% (v/v) Triton X-100. Lysates werecentrifuged at 15,000 rpm for 5 minutes at 4�C and thesupernatant added to 50mLof 50%(v/v)m7GTP-Sepharosebeads (GE Healthcare). Samples were incubated for 25minutes at 4�C on an Eppendorf tube shaker, after whichthe beads were washed 3 times in buffer A and isolated bycentrifugation. Bound protein was recovered by boiling thebeads for 5 minutes in SDS sample buffer and analyzed bySDS-PAGE.The following antibodies were used following the man-
Western blottingCLL cells were harvested and protein extracts made
using radioimmunoprecipitation assay (RIPA) buffersupplemented with protease and phosphatase inhibitors(Sigma). Proteins were separated using a 12% SDS-gel,and blotted onto a polyvinylidene difluoride (PVDF)membrane (Sigma). As part of this work, we comparedexpression of BCL2A1, BCL2L1, and MCL1, that is, themajor BCL2 family prosurvival proteins induced by stro-mal cell/CD154 culture in 36 patients. Densitometry datafromWestern blot analyses were controlled for differencesin loading using glyceraldehyde-3-phosphate dehydroge-nase (GAPDH), and a pool of cell lysates was included ineach gel to be able to compare autorads. Membranes wereprobed with BCL2L1 (1:1,000), phospho-AKT (1:1,000),and GAPDH (1:1,000; New England Biolabs). Anti-BCL2A1 serum was a gift from Dr. J. Borst (The Nether-lands Cancer Institute, Amsterdam, the Netherlands).Anti-phospho-4E-BP, phospho-S6 ribosomal protein,phospho-S6 kinase, and pospho-eIF4E were from NewEngland Biolabs and were used at 1:1,000. Anti-NOXA(Enzo Life Sciences) and anti-BIM (New England Biolabs)were also used at 1:1,000. Secondary antibodies–anti-mouse-immunoglobulin G (IgG)–horseradish peroxidase(HRP) or anti-rabbit-IgG-HRP (Sigma)—were used at1:2,000.
35S methionine incorporationCells (3 � 106/mL) were cultured on plastic or 80% to
90% confluent and 35 Gy irradiated stromal layers for 24hours. 35S methionine (3 mL of 37 MBq/mL; PerkinElmer)was added to each well and cells cultured for an additional
30 minutes at 37�C. After harvesting, cells were washed inPBS, lysed using Passive Lysis Buffer (Promega), centrifuged(13,000 rpm, 1 minute) to remove cellular debris, and theprotein was precipitated using 25% trichloro-acetic acid(Sigma). Lysates were transferred to filter papers using aMillipore vacuum manifold and incorporation of radioac-tivity measured using a Wallac liquid scintillation counter(PerkinElmer).
Cell viabilityCLL cells (5� 105/well of a 96-wellmicrotiter plate) were
cultured for 24 hours with 4EGI-1, thapsigargin, or ABT-737. Cells were harvested from the stromal layer and intra-cellular ATP was determined by incubation with CellTiter-Glo reagent (100 mL; Promega) for 10 minutes in opaqueplates before the luminescence intensity was read using aWallac Victor 1420 Multilabel counter.
Statistical analysisStatistical analyses were conducted using GraphPad
Prism version 4.0b (GraphPad Software Inc.). Pharmaco-logic analysis of drug effects, singly and in combinationwasconducted with CalcuSyn version 2 (Biosoft; ref. 30).
ResultsCulture on stromal cells in the presence and absence ofCD154 increases protein synthesis and cap-complexformation in a PI3Ka-dependent manner
35S methionine incorporation was used to determineeffects of culture conditions on overall protein synthesis.Stromal cell culture induced a 6-fold increase in 35Smethionine incorporation (paired t test; P ¼ 0.006) witha further increment due to the presence of CD154 (Fig.1A). To determine changes to cap-complex formationunder different culture conditions cap-binding assayswere conducted. There was little association of eIF4Gwith eIF4E in freshly isolated leukemic cells, but follow-ing culture on stromal cells alone the amount of eIF4Gincreased as compared with basal conditions, with stro-mal cell/CD154 culture producing a further increment(Fig. 1B). These results showed that induction of cap-complex formation associated with an increase in overallprotein synthesis and both were effects of culture withstromal cells/CD154.
PI3K signaling induced translation in normal and can-cer cells and is enhanced by stromal cell contact in CLL(31). We, therefore, determined the role of this pathwayin leukemic cells in stromal cell/CD154 culture. Thenonisoform-specific PI3K inhibitor, LY294002, reducedprotein synthesis by approximately 70% (Fig. 1C; pairedt test; P ¼ 0.007) and produced a corresponding reduc-tion in the association of eIF4G with eIF4E in cap-bindingassays (Fig. 1B). A specific PI3Ka inhibitor, PI-103,repressed protein synthesis (P ¼ 0.02), whereas a PI3Kdinhibitor, PIK-294, had no effect (Fig. 1C). In keepingwith this, PI-103 reduced cap-complex formation to sub-basal levels, whereas PIK-294 was ineffective (Fig. 1D).
Cap-Translation Inhibition in CLL
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Both rapamycin (P ¼ 0.007), an inhibitor of mTORC1,and the eIF4E/eIF4G interaction inhibitor, 4EGI-1 (P ¼0.004), repressed protein synthesis and cap-complexformation
4EBP1 and S6K are important targets of PI3K/AKT/mTOR in the control of cap-dependent translation.Stromal cell/CD154 culture induced 4EBP1 phosphory-lation (Fig. 2A and C) and this was abolished byLY294002, PI-103 and rapamycin, with LY294002 beingleast effective at the concentrations used. 4EBP1 phos-phorylation was not altered by 4EGI-1, as anticipatedfrom its mechanism of action, or by PIK-294, inkeeping with ineffectiveness of this agent in reducing35S methionine incorporation and cap-complex forma-tion (Fig. 2C). S6K seemed to be expressed at low levelsin CLL although some induction was seen on stromalcell/CD154 culture (Fig. 2A). Ribosomal protein S6(rpS6) is a direct target of S6K and its phosphorylationalso increased on stromal cell/CD154 culture and wassensitive to PI3K inhibition (Fig. 2B). Therefore, PI3K,which has previously been shown to be required forleukemic cell survival on stromal layers (31), and spe-cifically the PI3Ka isoform, is responsible for 4EBP1phosphorylation and regulates cap-binding complexformation in CLL.
Highly translated mRNAs are found on heavy poly-somes, whereas those that are not being translated arefound on lighter polysomes or monosomes. To assesspolysome formation, nuclear-poor cell lysates were sub-jected to sucrose density gradient centrifugation. Poly-somes were readily detectable in the human cell line,MCF7 (Supplementary Fig. S1) and in CLL cells increasedafter culture with stromal cells/CD154 from very lowlevels (Fig. 3A). Administration of rapamycin and 4EGI-1 reduced polysomes. As a measure of the change intranslation efficiency, we compared the area under thepolysome component of the trace (fractions 7 to 12) tothat under the monosome component (fractions 1 to 6;ref. 32). In stromal cell/CD154 conditions, the polysome:monosome ratio was 0.36, and this fell to 0.1 after admin-istration of rapamycin and 0.15 after 4EGI-1 (Fig. 3A)suggesting an overall reduction in translation efficiencydue to these inhibitors.
Phosphorylated eIF4E and 4EBP1 are found inproliferation centers
Proliferation centers in CLL lymph nodes are identi-fied as clusters of prolymphocytes and para-
Figure 1. Protein synthesis and cap-complex formation is increased by stromal cell/CD154 contact by a PI3Ka-dependent mechanism. A, 35Smethionine incorporation. CLL cells were cultured on plastic (PL), stromal cells alone (stroma), or stromal cells with CD154 (stroma/CD154). After24 hours, culture 35S methionine was added for 30 minutes and the cells harvested and lysed followed by measurement of incorporated radioactivity.Mean � SEM for 3 patients are presented. Protein synthesis is increased significantly by stromal cell contact (paired t test; P ¼ 0.006) andstromal cell/CD154 culture (P ¼ 0.017). B, cap-binding assays. Lysates from freshly isolated cells, and cells cultured on stromal cells (NT) or stromalcells with CD154 (CD154) in the presence or absence of LY294002, were mixed with m7-GTP beads. Western blot analyses of material from thewashed beads were probed with anti-eIF4E and anti-eIF4G antibodies. Representative of 4 patients. C, effects of PI3K inhibitors, LY294002 and PI-103,and 4EGI-1 on 35S methionine incorporation. Mean � SEM are presented for 3 patients. As compared with stromal cell/CD154 culture withoutinhibitor protein synthesis is significantly reduced by LY294002 (paired t test; P ¼ 0.007), PI-103 (P ¼ 0.02), and 4EGI-1 (P ¼ 0.004). D, cap-bindingassays to show the effects on cap-complex formation of a PI3Ka-specific inhibitor, PI-103, a PI3Kd specific inhibitor, PIK-294, an mTORC1 inhibitor,rapamycin, and an inhibitor of the interaction between eIF4E and eIF4G. Representative of 4 patients.
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immunoblasts with a higher fraction of cells expres-sing the proliferation marker, Ki-67 than in the sur-rounding tissue. Reasoning that proliferation centers arelikely to be sites of increased translation in leukemiccells in vivo, we looked for evidence of increased ex-
pression of phosphorylated eIF4E and 4EBP1 in CLLlymph node sections. Immunohistochemistry, reveal-ed staining in proliferation centers (Fig. 3B and C)suggesting increased translation in these structuresin vivo.
Figure 2. Stromal cell/CD154 contact is sufficient to induce 4EBP1 phosphorylation. A, Western blot analysis showing changes in expression ofphosphorylated S6K and 4EBP1 on stromal cell/CD154 culture in the presence of LY294002, PI-103, or 4EGI-1. Gray and black arrowheadsindicate isoforms of 4EBP1 and GAPDH is a loading control. B, Western blot analysis showing changes in phosphorylation of rpS6 with stromalcell/CD154 culture in the presence and absence of inhibitors, LY294002, PI-103, and 4EGI-1. C, Western blot analyses showing change in4EBP1 phosphorylation in the presence of the PI3Ka-specific inhibitor, PI-103, and the PI3Kd inhibitor, PIK-294. The gray arrowhead indicates thepredominant isoform in basal conditions and the black arrowhead the isoform induced by stromal cell/CD154 culture. GAPDH is a loadingcontrol. Western blot analyses are representative of 4 patients.
Figure 3. Stromal cells/CD154culture increases polysomeformation. A, spectrophotometrictraces of fractions taken fromsucrose density gradients of nuclear-poor lysates from CLL cells. Tracesare from freshly isolated leukemiccells (PB), following 24-hour cultureon stromal cell/CD154 and followingstromal cell/CD154 culture in thepresence of rapamycin or 4EGI-1immunohistochemistry ofproliferation centers stained with (B)anti-phospho-eIF4E and (C) anti-phospho-4EBP1. The magnificationis �40.
Cap-Translation Inhibition in CLL
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Partial dissociation of BCL2A1, BCL2L1, and MCL1frompolysomes following inhibitionof cap-dependenttranslation
mRNAs differ in their sensitivity to inhibition of cap-dependent translation (33). To determine whether inhibi-tion of cap-complex assembly reduced association of spe-cific mRNAs with polysomes, we measured amounts ofmRNA in fractions of cell lysates separated on sucrosedensity gradients.RPS6mRNAwas substantially dissociatedfrom polysomes after administration of LY294002 [82% �
10% in fractions 7–11 without inhibitor to 32%� 8%withinhibitor (mean � range), n ¼ 3] but actin (ACTB) mRNAwas relatively resistant to this agent (78% � 11% withoutinhibitor to 62%� 9%; Fig. 4A). To show that these effectswere specific, we used EDTA to dissociate the ribosomalsubunits and found 35%� 7% of ACTBmRNA and 33%�8% of RPS6 mRNA in fractions 7 to 11 after treatment(Fig. 4A). We, therefore, distinguish mRNAs that are highlysensitive to inhibition of cap-dependent translation (RPS6)from those, such as actin, that are less sensitive (33).
Figure 4. 4EGI-1 partially reduces polysome association and inhibits protein expression of BCL2L1 and BCL2A1. A, nuclear-poor lysates made after 24 hoursof stromal cell/CD154 culture in the presence (gray shaded area) and absence (dotted line) of LY294002or EDTAwere separatedon sucrose density gradients.Fractions were removed and RPS6 or actin (ACTB) mRNA measured by real-time semi-quantitative PCR. The amount of mRNA in each fraction ispresented as a percentage of the total mRNA. Representative of 3 patients. B, nuclear-poor lysates produced from CLL cells after 24 hours of stromalcell/CD154 culture in the presence (gray shaded area) or absence (dotted line) of 4EGI-1 or LY294002were separated on sucrose density gradients. Fractionswere removed and mRNA for BCL2A1, BCL2L1, and MCL1 was measured by real-time semi-quantitative PCR. The amount of mRNA in each fraction ispresented as a percentage of the total mRNA (representative of 6 patients). C, Western blot analyses from 2 patients (left hand panels #38 and righthand panels #13; Table 1) showing changes in protein expression after 24 hours of stromal cell/CD154 culture in the absence and presence of LY294002,PI-103, rapamycin, and 4EGI-1. GAPDH is a loading control. D, densitometry of Western blot analyses of BCL2 family survival proteins (n ¼ 6). Theoverall amounts of the BCL2 family survival proteins are significantly reduced following administration of 4EGI-1 (P ¼ 0.0013, paired t test). This differenceis due to reduction in BCL2A1 (P < 0.0001) and BCL2L1 (P ¼ 0.0086) but not MCL1.
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We determined the sensitivity of BCL2 family prosur-vival proteins most highly expressed in the lymph nodemicroenvironment–BCL2A1, BCL2L1, and MCL1–toinhibition of cap-dependent translation. Administrationof 4EGI-1 (ref. 28; Fig. 4B) caused the amount of BCL2A1mRNA associating with the polysomal fractions (fractions7–11) to decrease from a mean of 74% to 50% (n ¼ 3).Similarly, BCL2L1 mRNA decreased from 86% to 60%and MCL1 mRNA from 57% to 44%. To determinewhether these effects were dependent on the inhibitorused, we repeated the experiments with LY294002 andsimilarly found partial dissociation of mRNA from poly-somes (Fig. 4B).To determine the significance of these changes for pro-
tein expression, we conducted Western blot analysis for agroup of 6 patients (Fig. 4C and Supplementary Fig. S2A).4EGI-1 repressed both BCL2A1 (P < 0.0001, paired t test)and BCL2L1 (P ¼ 0.0086) expression but not MCL1,whereas LY294002, PI-103, and rapamycin produced littlerepression of any of these prosurvival proteins (Fig. 4C).MCL1 protein stability was not diminished following
treatment with 4EGI-1 suggesting that there is unlikely tobe a major effect of 4EGI-1 on degradation of this protein(Supplementary Fig. S2B).In summary, LY294002, PI-103 and rapamycin caused
partial dissociation of BCL2A1, BCL2L1, and MCL1mRNAs from polysomes but this was not sufficient torepress protein expression. 4EGI-1 produced a differentpattern of protein responses with repression of BCL2A1and BCL2L1 but not MCL1 (Fig. 4D), whereas causingsimilar changes to the polysome profiles of BCL2A1,BCL2L1, and MCL1 as LY294002. This suggests that4EGI-1 has mechanisms of action in addition to inhibitionof cap-complex formation.
4EGI-1 induces an ER stress responseWork by others suggests that 4EGI-1 might induce com-
ponents of an ER stress response (34). ATF4 is such acomponent and forms part of a protein complex thatinduces proapoptotic, NOXA (35). 4EGI-1 induced ATF4and NOXA in myeloma (34). We determined whether4EGI-1 induced ER stress response proteins in CLL.Westernblot analysis showed that ATF4 and CHOP (Fig. 5A) wereinduced by tunicamycin and thapsigargin, known inducersof ER stress responses, and by 4EGI-1 but not by LY294002,PI-103, and rapamycin (Fig. 5B). MCL1, BCL2A1, andBCL2L1 expression was maintained following thapsigargintreatment (Fig. 5C) suggesting that induction of an ER stressresponse alone was not sufficient to alter expression ofthese proteins. Induction of phosphorylated eIF2a isresponsible for translation inhibition produced by ER stressresponses and 4EGI-1 produced a modest increase in thisprotein that was not observed with LY294002 or rapamycin(Fig. 5D). To show an effect of phosphorylated eIF2a onBCL2A1, BCL2L1, andMCL1, we used, salubrinal, a specificinhibitor of eIF2a dephosphorylation (ref. 29; Fig. 5E).Salubrinal repressed BCL2L1 and BCL2A1 but not MCL1suggesting that BCL2L1 and BCL2A1 weremore sensitive to
the inhibitory effects of phosphorylated eIF2a than MCL1.Overall, neither an ER stress response alone (thapsigargin)nor repression of cap-dependent translation alone(LY294002 or rapamycin) was sufficient to repress BCL2A1or BCL2L1. However, 4EGI-1, which both repressed cap-dependent translation and induced ER stress response pro-teins including ATF4, did accomplish these effects.
BH3-only protein, NOXA, is induced by 4EGI-14EGI-1 induced NOXA in CLL (n ¼ 6; Fig. 5F) and
densitometry showed that the NOXA:MCL1 ratioincreased from 0.9 to 5.2 (Fig. 5G; ref. 7). ProapoptoticBIM also binds MCL1 and stromal cell/CD154 culturecaused appearance of a slower migrating form of BIMsuggesting phosphorylation (36). The addition of 4EGI-1caused a decrease in expression of the high molecularweight BIM isoform, BIMEL, but no change to amounts ofBIML (Supplementary Fig. S2C). Overall, 4EGI-1 stronglyinduced NOXA in CLL cells.
4EGI-1 and ABT-737 synergize to reduce cell viability4EGI-1 reduced amounts of prosurvival proteins (Fig.
4C) in leukemic cells supported on stromal cells/CD154and induced proapoptotic NOXA (Fig. 5F), the latter aspart of an ER stress response. This suggested that 4EGI-1might reduce viability of CLL cells cultured in this systemand accordingly we determined its effects in a groupof unselected patients with CLL (n ¼ 11). 4EGI-1 at 100mmol/L, the maximum concentration used, reduced viabil-ity by approximately 63% (Fig. 6A). To determine thecontribution of ER stress response to reducing cell viability,we used thapsigargin (Fig. 5C). This agent achieved areduction in viability of only approximately 25%, at bothits maximum (5 mmol/L) concentration and 50% maximalconcentration, suggesting that inhibition of the ER stressresponse was not sufficient to reproduce the effects of 4EGI-1. Leukemic cells cultured on stromal cells/CD154 arerelatively resistant to the BCL2/BCL2L1 antagonist ABT-737 (4) but by combining ABT-737 with thapsigargin areduction in viability similar to that of 4EGI-1 alone wasachieved. This result suggested that neither induction ofER stress nor inhibition of BCL2/BCL2L1 were alone suf-ficient to reproduce the effects of 4EGI-1, but togetherthapsigargin and ABT-737 produced a similar decrease inviability to that observed with 4EGI-1. The combination of4EGI-1 and ABT-737 produced further enhancement withviability reduced by approximately 90% suggesting thatcell survival in the presence of 4EGI-1 was due to residualBCL2/BCL2L1 expression and that 4EGI-1 can overcomeresistance to ABT-737.
To formally determine synergy, thapsigargin and 4EGI-1were used in a fixed ratio with ABT-737 at 4 concentrations.For both thapsigargin/ABT-737 and 4EGI-1/ABT-737, thecombination index (CI) was less than 1 (Fig. 6B) indicatingsynergy (30).
We considered that higher amounts of MCL1 (37) andother BCL2 family prosurvivalmembers inducedby stromalcell/CD154 contact might be factors reducing effectiveness
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of combined 4EGI-1 andABT-737.We, therefore, comparedrelative expression of BCL2A1, BCL2L1, and MCL1 in 36patients (Supplementary Fig. S3) showing a range of expres-sion levels. The combination of 4EGI-1 and ABT-737 waseffective in patients with either low or high relative BCL2family prosurvival protein expression, with lack of enhance-ment being observed in only 1 patient. Our results suggestthat specific translation inhibitors synergize with ABT-737to overcome resistance to apoptosis caused by stromalcell/CD154 culture.
DiscussionCLL cells within the lymph node microenvironment
show increased proliferation as compared with those in
the peripheral circulation (38) and are also likely to receivesignals from T cells. Recent work has shown that in vitroresponses to stimulation by the T-cell surface marker,CD154, are associated with clinical outcome (39). Leuke-mic cells that proliferate within the lymph node microen-vironment are a target for therapy, which has promptedwork to understand signaling pathways and mechanismsof survival in this context (11) and, in turn, this hasrequired the development of specialized culture systems(40–43).
We showed for the first time that cap-complex formationand overall protein synthesis is induced by contact ofleukemic cells with a fibroblast cell layer with a furtherincrement produced by the addition of CD154. We used
Figure 5. 4EGI-1 produces an ER stress response with induction of NOXA. Leukemic cells were either lysed without a period of culture (�) or cultured for 24hours in the presence of stromal cells/CD154 alone (þ) or in the presence of the indicated inhibitor. A, 4EGI-1 induces ER stress response proteins.Western blot analyses showing the effects of 4EGI-1 (50 and 100 mmol/L), tunicamycin (Tu; 2.5 and 5 mmol/L), and thapsigargin (Tg; 2.5 and 5 mmol/L) onexpressionof ATF4andCHOP.B,ERstressproteins arenot inducedbyPI3K inhibitors, LY294002 (10 and20mmol/L), PI-103 (0.2 and1mmol/L), andmTORC1inhibitor, rapamycin (Rapa; 20 and 50 ng/mL). Western blot analyses for ATF4 and CHOP are shown. C, Western blot analyses showing effects of ER stressinducer thapsigargin at 2.5 and 5 mmol/L on BCL2 family proteins NOXA, MCL1, BCL2L1, and BCL2A1. (�) indicates lysates from cells freshly isolatedfrom patients and (þ) after 24 hours culture on stromal cells/CD154. Both sections of the panel are from the same autorad. GAPDH was used as aloading control. D,Western blot analysis showingphosphorylated eIF2aexpression in response to LY294002, rapamycin, and4EGI-1. Total eIF2a is usedasaloading control. E, BCL2L1, BCL2A1, and MCL1 expression following administration of salubrinal (100 mmol/L). F, Western blot analysis of 3 patients(#38, #39, #40; Table 1) showing MCL1 and NOXA expression following stromal cell/CD154 culture in the presence and absence of 4EGI-1. GAPDH is aloading control. G, densitometry of Western blot analysis was used to derive values for NOXA/GAPDH (N) and MCL1/GAPDH (M). Normalized expression isshown for freshly isolated cells (�), cells cultured on stromal cells/CD154 (CD154), and cells cultured on stromal cells/CD154 with 4EGI-1 (CD154þ 4EGI-1).Mean � SEM. For each experimental condition the NOXA:MCL1 ratios are presented above the bracket.
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PI3K and mTORC1 inhibition to reduce cap-complex for-mation but showed that these agents were not effective inrepressing BCL2L1, BCL2A1, or MCL1 protein expression.We found relatively little dissociation of ACTBmRNA frompolysomes, which is consistent with work from others (33)showing lack of sensitivity of ACTB mRNA to overexpres-sion of eIF4E. Our results suggested that the specific BCL2family prosurvival gene mRNAs we analyzed behaved in asimilarmanner toACTB andwere relatively little dissociatedfrom polysomes by PI3K inhibition. Therefore, despiterepression of cap-dependent translation, there was continu-ing expression of BCL2L1, BCL2A1, and MCL1 proteins.BCL2L1 has a 50-untranslated region (UTR0 of 376 bp andcontains an internal ribosome entry sequence site (44)providing a mechanism for continuing translation despiterepression of cap-dependent translation but MCL1 andBCL2A1 have shorter 50-UTRs at approximately 50 bp.Further work is needed to elucidate the mechanism forcontinued expression ofMCL1 and BCL2A1 in the presenceof inhibitors of cap-dependent translation but the simplestexplanation is continuing low-level cap-dependent transla-tion, which is sufficient for MCL1 and BCL2A1 expression.Apoptosis causes inhibition of protein synthesis (33) andspeculatively BCL2 family prosurvival protein levels could
be relatively resistant to alterations in cap-complex forma-tion to maintain cell survival.
In contrast with PI3K and mTORC1 inhibition, 4EGI-1repressed protein expression of BCL2L1 and BCL2A1 sug-gesting mechanisms of action in addition to repression ofcap-dependent translation.
Resistance to the effects of small-molecule BH3mimeticsis a clinical problem limiting the effectiveness of theseagents. ABT-737 has a similar action to the BH3-onlyprotein, BAD, and is a BCL2 and BCL2L1 antagonist. It hasbeen suggested that ABT-737 can be used as a probe todissect the BCL2 family protein interactions important forsurvival (45). Freshly isolated CLL cells are very sensitive toABT-737, but this was significantly diminished when theleukemic cells were cultured with stromal cells and CD154(4, 7). The implication was that that freshly isolated leu-kemic cells were dependent on BCL2 (BCL2L1 not beingexpressed in these conditions; ref. 46), but on culture withstromal cells/CD154 survival became dependent on MCL1and BCL2A1 (5, 7). There are, therefore, 2 possible ways tosensitize leukemic cells on stromal cell/CD154 culture toABT-737: first, repression of BCL2A1 or MCL1, and second,induction of BH3-only proteins, especially NOXA, whichbinds MCL1 and BCL2A1 (47). Work by others has shown
Figure 6. 4EGI-1 enhances apoptosis due to ABT-737 across a range of different BCL2A1, BCL2L1, and MCL1 expression levels. A, dose-effectcurves for ABT-737, 4EGI-1, and thapsigargin (Tg) alone and in combination (n ¼ 11). Viability was determined by luminescent detection of ATP(CellTiter-Glo; Promega) at 4 different drug concentrations. For 100% doses, ABT-737 was used at 1 mmol/L, 4EGI-1 at 100 mmol/L, and thapsigargin at5 mmol/L. B, synergy was determined by CI-fractional effect analysis (Calcusyn; Biosoft). CI < 1 indicates synergy. C, diagram showing possiblemechanism of action of 4EGI-1. Cap-binding assays show that LY294002, PI-103, and rapamycin repress cap-complex formation in CLL cells onstromal cell/CD154 culture but do not alter BCL2L1, BCL2A1, or MCL1 protein expression. 4EGI-1 inhibits cap-dependent translation and also inducesNOXA through an ER stress response to antagonize the effects of MCL1. Inhibition of translation by phosphorylated eIF2a expression, whoseexpression is maintained by 4EGI-1 in contrast to LY294002 or rapamycin, together with inhibition of cap-dependent translation may be necessary forreduction in BCL2A1 and BCL2L1 expression.
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that knockdown of NOXA enhances resistance to ABT-737in primary B cells and induction of NOXA by fludarabinesynergizes with ABT-737 in CLL (7).
These considerations prompted analysis of NOXA ex-pression in response to 4EGI-1. We showed inductionof this protein while amounts of BIM, which binds BCL2,BCL2L1, MCL1, and BCL2A1, remained essentially un-changed, although some repression of the BIMEL isoformwas observed, there was no change to the more apoptoticBIML and BIMS. Overall, the 4EGI-1 produced a highlyfavorable pattern of responses for enhancement of theeffects of ABT-737, and we showed synergy between theseagents in patients expressing varying amounts of MCL1.Thapsigargin induced NOXA but did not reproduce theeffects of 4EGI-1 on cell viability and our overall view isthat induction of NOXA by 4EGI-1 is necessary but notsufficient for its effects.
NOXA induction by 4EGI-1 has previously beenreported in myeloma cell lines and primary cells (34)and we suggest that this may prove to be a predictablesecondary effect of using this agent. NOXA has also beeninduced by a variety of other agents in association with ERstress (48). Induction of ATF4 in an ER stress response(49) involves skipping of upstream open reading framesmediated by the translation inhibitor, phosphorylatedeIF2a. Induction of ATF4 in CLL and myeloma by4EGI-1 (34) provides indirect evidence for the functionalimportance of phosphorylated eIF2a. In addition, com-bining thapsigargin and ABT-737 reduced CLL viability toa similar level to 4EGI-1 (Fig. 6A) suggesting its effectsrequired both inhibition of cap-dependent translationand induction of an ER stress response. ER stress issufficient to cause CLL apoptosis when cultured on plastic(50) but in stromal cell/CD154 culture thapsigargin didnot repress BCL2 family prosurvival proteins (Fig. 5C) orproduce major loss of cell viability (Fig. 6A). We speculatethat the combination of partial repression of cap-depen-dent translation together with an ER stress response, that
is, translation inhibition by phosphorylated eIF2a andinduction of NOXA, was required for the observed effectsof 4EGI-1 (Fig. 6C).
In this report, we showed first that cap-dependenttranslation is induced by a culture system mimickingthe lymph node microenvironment, and second that4EGI-1 reduced cap-complex formation and inducedan ER stress response. 4EGI-1 reduced levels of prosur-vival BCL2L1 and BCL2A1 and induced NOXA creatinga favorable situation for synergy with ABT-737. Thiscombination of agents may be the basis for a therapeu-tically useful approach to target leukemic cells resistantto conventional chemotherapy in the lymph nodemicroenvironment.
Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.
Authors' ContributionsConception and design: S. Willimott, D. Beck, S.D. WagnerDevelopment of methodology: S. Willimott, M.J. AhearneAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): S. Willimott, D. Beck, M.J. Ahearne, V.C. AdamsAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): S. Willimott, D. Beck, S.D. WagnerWriting, review, and/or revision of the manuscript: M.J. Ahearne, S.D.WagnerStudy supervision: S.D. Wagner
AcknowledgmentsThe authors thank the patients for their contribution to this study.
Grant SupportThis work was funded by a grant to S.D. Wagner from the Kay Kendall
Leukemia Fund.The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.
Received June 29, 2012; revised March 19, 2013; accepted April 12, 2013;published OnlineFirst April 30, 2013.
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2013;19:3212-3223. Published OnlineFirst April 30, 2013.Clin Cancer Res Shaun Willimott, Daniel Beck, Matthew J. Ahearne, et al. in Chronic Lymphocytic LeukemiaApoptosis through Cap-Dependent and -Independent Mechanisms Cap-Translation Inhibitor, 4EGI-1, Restores Sensitivity to ABT-737
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