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apeutics, Targets, and Chemical Biology

ivity of the Novel Dual Phosphatidylinositol 3-Kinase/malian Target of Rapamycin Inhibitor NVP-BEZ235

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inst T-Cell Acute Lymphoblastic Leukemia

esca Chiarini1, Cecilia Grimaldi1, Francesca Ricci3, Pier Luigi Tazzari3, Camilla Evangelisti1,
a Ognibene4, Michela Battistelli6, Elisabetta Falcieri5,6, Fraia Melchionda2, Andrea Pession2, alepaolo Pagliaro3, James A. McCubrey7, and Alberto M. Martelli1,5
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ent findings have highlighted that constitutively active phosphatidylinositol 3-kinase (PI3K)/Akt/alian target of rapamycin (mTOR) signaling is a common feature of T-cell acute lymphoblastic leu-(T-ALL), where it upregulates cell proliferation, survival, and drug resistance. These observations lendlling weight to the application of PI3K/Akt/mTOR inhibitors in the therapy of T-ALL. Here, we haveed the therapeutic potential of the novel dual PI3K/mTOR inhibitor NVP-BEZ235, an orally bioavailablezoquinoline derivative, which has entered clinical trials for solid tumors, on both T-ALL cell linestient samples. NVP-BEZ235 was cytotoxic to a panel of T-ALL cell lines as determined by MTT assays.EZ235 treatment resulted in cell cycle arrest and apoptosis. Western blots showed a dose- and time-dent dephosphorylation of Akt and mTORC1 downstream targets in response to NVP-BEZ235. Remark-VP-BEZ235 targeted the side population of both T-ALL cell lines and patient lymphoblasts, which mightpond to leukemia-initiating cells, and synergized with chemotherapeutic agents (cyclophosphamide,bine, dexamethasone) currently used for treating T-ALL patients. NVP-BEZ235 reduced chemoresistancecristine induced in Jurkat cells by coculturing with MS-5 stromal cells, which mimic the bone marrowenvironment. NVP-BEZ235 was cytotoxic to T-ALL patient lymphoblasts displaying pathway activation,the drug dephosphorylated eukaryotic initiation factor 4E-binding protein 1, at variance with rapamy-aken together, our findings indicate that longitudinal inhibition at two nodes of the PI3K/Akt/mTORrk with NVP-BEZ235, either alone or in combination with chemotherapeutic drugs, may be an efficient
netwo
treatment of those T-ALLs that have aberrant upregulation of this signaling pathway for their proliferationand survival. Cancer Res; 70(20); 8097–107. ©2010 AACR.

and aadultimproT-ALLactiva

duction

ll acute lymphoblastic leukemia (T-ALL) is an aggres-isorder of precursor cells committed to the T-cell line-). Over the past 20 years, survival rates of T-ALL

proved, mainly because of advances in che-ocols. Survival rates at 5 years for children

survivway i(PI3K)ing neseveraHES1al supon choxidatstrom(10). AAkt/mtwo cand minhibiloguespromi

ns: 1Department of Human Anatomy and 2Paediatricaematology Unit “Lalla Seràgnoli,” University ofhaematology and Transfusion Center, Policlinico S.4Musculoskeletal Cell Biology Laboratory, I.O.R.;ne di Bologna c/o I.O.R., Bologna, Italy; 6DISUAN,ino “Carlo Bo,” Urbino, Italy; and 7Department ofImmunology, School of Medicine, East Carolinalle, North Carolina

tary data for this article are available at Cancerttp://cancerres.aacrjournals.org/).

uthor: Alberto M. Martelli, Dipartimento di Scienzee, Università di Bologna, 40126 Bologna, Italy. Phone:ax: 39-051-2091695; E-mail: [email protected].

5472.CAN-10-1814

ssociation for Cancer Research.

ls.org

Research. on October 19, 20cancerres.aacrjournals.org ed from

dolescents with T-ALL are 70% to 75%, whereas fors, the rates are 35% to 40% (2). In spite of thesevements, novel and less toxic treatment strategies forare needed (3). Novel therapies may target aberrantlyted signaling pathways influencing the proliferation,al, and drug resistance of these T-ALLs. One such path-s represented by the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (mTOR) signal-twork (4, 5). Pathway upregulation in T-ALL is due tol reasons, which include Notch1 activation leading to(hairy and enhancer of split 1)-mediated transcription-pression of the PTEN (phosphatase and tensin deletedromosome 10) gene (6, 7), PTEN phosphorylation orion (8), interleukin (IL)-4 secreted by bone marrowal cells (9), or mutations affecting PI3K, PTEN, or Aktbout 85% of T-ALL patients display increased PI3K/TOR activation at diagnosis (8, 11). mTOR exists asomplexes, referred to as mTOR complex 1 (mTORC1)TOR complex 2 (mTORC2; ref. 12). Allosteric mTORtors, which include rapamycin and its analogues (rapa-
), mainly target mTORC1. These inhibitors displaysing effects in preclinical models of T-ALL (13, 14).
8097

20. © 2010 American Association for Cancer

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ver, rapamycin/rapalogues are mainly cytostatic (15)uld hyperactivate Akt due to the existence of feedbackbetween mTORC1, PI3K, and Akt (16). Moreover, it isning to emerge that there are mTORC1 functions,ing phosphorylation of eukaryotic initiation factording protein 1 (4E-BP1; which controls translation),are insensitive to rapamycin/rapalogues (17, 18). Re-, dual PI3K/mTOR inhibitors have been synthesized,, unlike rapamycin/rapalogues, target the catalytic siteh kinases (19). As such, these compounds are pro-otic and dephosphorylate 4E-BP1. We have recentlythe cytotoxic effect of the dual PI3K/mTOR inhibitorin preclinical settings of T-ALL (20). PI-103 has proveny also in vivo against models of human tumors xeno-in mice, where it displayed low toxicity and was well

ted (19). However, PI-103 did not enter clinical trials,y because of its rapid in vivo metabolism (21). Here,ve analyzed the therapeutic potential of the novel dualmTOR inhibitor NVP-BEZ235, an orally bioavailableoquinoline derivative (22), which has entered clinicalfor solid tumors, in both T-ALL cell lines and patientes. We have shown that this drug displayed strongxic activity against T-ALL cells. Rapid commitmentLL cells to death was triggered by NVP-BEZ235. Impor-, healthy donor peripheral blood CD4+ T cells wereless sensitive than the T-ALL cell lines tested, suggest-favorable therapeutic index. Combinations of NVP-5 with conventional anti–T-ALL chemotherapeuticshowed a strong synergistic activity, implying thatNVP-BEZ235–based combinations could be feasibleclinic. Thus, our results provide the framework for
al trials of the dual PI3K/mTOR inhibitor NVP-
WesteThi

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rials and Methods

ials-BEZ235 was kindly provided by Novartis. For Westerng, primary antibodies were purchased from Cell Sig-Technology. Rapamycin, vincristine, dexamethasone,hosphamide, cytarabine (Ara-C), Hoechst 33342, andemorgin C were purchased from Sigma-Aldrich. PI-103rchased fromAlexis Biochemicals. KO143was purchasedxon Medchem BV. Antibody to ABCG2 was from Milli-pstate, antibody to 170-kDa P-glycoprotein (P-gp) wasamiya, and antibody to MRP1 was from BD Biosciencesingen. AlexaFluor-conjugated antibodies to PTEN,p-Akt, Thr37/46 p-4E-BP1, and Ser235/236 p-S6 ribo-protein (p-S6RP) were from Cell Signaling Technology.

ulture and primary samplesT-ALL cell lines Jurkat, MOLT-4, CEM-S, CEM-R (CEM0, drug-resistant cells overexpressing P-gp; ref. 23),8402, and BE-13 were grown in RPMI 1640 supple-d with 10% fetal bovine serum (FBS). All cell lines
rom Deutsche Sammlung von Mikroorganismen undlturen GmbH and were characterized as specified
synerg>1.1 a

r Res; 70(20) October 15, 2010

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//www.dsmz.de/human_and_animal_cell_lines/main.ontentleft_id=21). Patient samples or peripheral bloodT lymphocytes from healthy donors were obtainednformed consent according to institutional guidelinesolated using Ficoll-Paque (Amersham Biosciences) fort lymphoblasts or by magnetic labeling (Miltenyi Bio-r CD4+ T lymphocytes. CD4+ T lymphocytes (105 perwere cultured in RPMI 1640 containing 10% FBS andlated for 24 hours with a mixture of 10 μg/mL phyto-glutinin-M (PHA-M) and 50 ng/mL human recombi-L-2 to induce proliferation.

iability analysisT assays were performed to assess the sensitivity ofo drugs, as previously described (24).

in V-FITC/propidium iodide stainingdetermine the extent of apoptosis induction, flow cyto-analysis of Annexin V-FITC/propidium iodide (PI)–

d samples was performed (24). Samples were analyzedEPICS XL flow cytometer (Beckman Coulter) with thepriate software (System II, Beckman Coulter). At leastevents per sample were acquired.

ycle analysiscytometric analysis was performed using PI/RNase A

ng according to standard procedures, as describedusly (24).

mission electron microscopys was performed as described previously (25).

rn blot analysiss was performed by standard methods, as previouslyed (24). Analysis with an antibody to β-actin showedprotein loading.

ture with mouse MS-5 stromal cellsat cells (1 × 106/mL) were seeded on top of MS-5stromal cells (at ∼70% confluence) for 3 hours beforedition of NVP-BEZ235 alone, vincristine alone, or anation of NVP-BEZ235 and vincristine. After 24 hoursubation at 37°C, cells were harvested with trypsin/, washed, and resuspended in binding buffer containingin V-FITC. Cells were counterstained with a phycoery-PE)-conjugated anti-CD45 antibody or with an irrelevantic control antibody and analyzed by flow cytometry afternic gating on CD45+ leukemia cells (26).

ined drug effect analysiscombination effect and a potential synergy were eval-from quantitative analysis of dose-effect relationshipscribed previously (24). For each combination experi-a combination index (CI) number was calculated usingosoft CalcuSyn software. This method of analysis gen-defines CI values of 0.9 to 1.1 as additive, 0.3 to 0.9 as
istic, and <0.3 as strongly synergistic, whereas valuesre considered antagonistic.
Cancer Research



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cytometric analysis of PTEN, p-Akt (Ser473),BP1 (Thr37/46), and p-S6RP (Ser235/236) levels-5 cells or T-ALL patient samples5 cells or lymphoblasts from pediatric patients withwere fixed in reagent 1 of the Intraprep Kit (Beckmanr) and permeabilized with saponin-based reagent 2, ased elsewhere (20). Cells were incubated with primarydies conjugated to AlexaFluor 488 or AlexaFluor 647.bit IgG conjugated to AlexaFluor 488 or AlexaFluoras used as an irrelevant antibody. Cells were analyzedC500 flow cytometer (Beckman Coulter). At least 5,000per sample were acquired.

cytometric detection of T-ALL sideation cells
s were resuspended at 1 × 106/mL in prewarmed ABC
n of caspase-8, caspase-9, and caspase-3 byNVP-BEZ235 (200 nmol/L). Cells wereifty micrograms of each lysate were electrophoresed on SDS-PAGE gels followed b

acrjournals.org


final concentration of 5 μg/mL in the presence orbsence of KO143 (0.1 μmol/L) or fumitremorgin Cmol/L) and the cells were incubated at 37°C forinutes with intermittent shaking. In some experi-, cells were stained with monoclonal antibodies tomembrane transporters (P-gp, MRP1, and ABCG2),ed by a PE-conjugated secondary antibody. Controles were analyzed with an irrelevant isotypic PE-gated antibody. Cells were then washed with PBSining 2% bovine serum albumin and processedoechst 33342 staining. Samples were analyzed withLab Quanta SC (Beckman Coulter) flow cytometer

ped with UV lamp and 488 solid state laser. Thest 33342 dye was excited at 366 nm. Side populationells were gated by the FL1/FL3 histogram, whereas
transporter staining was evaluated on the FL2
1640 with 2% FBS. Hoechst 33342 dye was added channel (27, 28).

1. NVP-BEZ235 induces cytotoxicity and apoptosis in T-ALL cell lines. A, MTT assays of T-ALL cell lines treated with NVP-BEZ235 for 24 h.parison between NVP-BEZ235 and PI-103. The results of MTT assays performed at 24 h are shown. Points and columns, mean of at least threet experiments; bars, SD. *, P < 0.01. C, flow cytometric analysis of Annexin V-FITC/PI–stained T-ALL cells treated with increasing NVP-BEZ235trations. The percentages of early apoptotic cells (Annexin-V FITC+/PI−; bottom right quadrant) and late apoptotic/necrotic cells (Annexin-V FITC+/PI+; topadrant) are indicated. The histograms are representative of three separate experiments performed in duplicate. D, Western blot analysis documenting

treatedwithNVP-BEZ235 for the indicated times, collected, and theny transfer onto a nitrocellulose membrane. CTRL, untreated sample.

Cancer Res; 70(20) October 15, 2010 8099



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EZ235 has cytotoxic proapoptotic effects oncell lineseffects of NVP-BEZ235 on T-ALL cells were analyzed byeating the cells with increasing concentrations of thend analyzing the rates of survival by MTT assays. Mostes displayed an IC50 for NVP-BEZ235 ranging from 80 tool/L, whereas the IC50 for BE-13 cells (which do not

y p-Akt; see following discussion) was 1 μmol/L. Theesistant CEM-R cell line had the highest sensitivity toEZ235 (IC50, 80 nmol/L; Fig. 1A). Next, a comparisonade between NVP-BEZ235 and PI-103, another dualmTOR inhibitor. When used at equimolar concentra-PI-103 was less effective than NVP-BEZ235 in affectingcell line growth (Fig. 1B) at all the concentrations tested.effects of NVP-BEZ235 on the induction of apoptosisetermined with Annexin V-FITC/PI staining in MOLT-at, and CEM-R cells. After 6 hours of NVP-BEZ235ent, flow cytometry analysis documented an increaseptosis in all the cell lines analyzed (Fig. 1C). Western
g analysis of extracts from MOLT-4, Jurkat, and CEM-treated with NVP-BEZ235 for 1, 3, and 6 hours showed
plus Itration

r Res; 70(20) October 15, 2010


ge of procaspase-8, procaspase-9, and procaspase-3D).

EZ235 blocks cells in the G0/G1 phase ofll cycleen the fundamental role played by PI3K/Akt/mTORling in cell proliferation (29), the effects of NVP-5 on cell cycle progression were also investigated.cytometric analysis of PI-stained T-ALL cells treatedVP-BEZ235 for 16 hours documented an increase inphase cells and a concomitant decrease in S andphases in both MOLT-4 and CEM-R cell lines (Supple-ry Fig. S1A). As pRb is a critical regulator of the celltransition from G1 to S phase (30), its phosphorylationwas analyzed by Western blotting. We detected ase in the amount of Ser807/811 pRb in MOLT-4 andR cells treated with 200 nmol/L NVP-BEZ235 forurs, whereas total pRb levels remained unchangedlementary Fig. S1A).ontrast, CD4+ T lymphocytes isolated from the periph-lood of healthy donors and stimulated with PHA-M
L-2 were much less sensitive to NVP-BEZ235 concen-s up to 500 nmol/L. The analysis, carried out by means
Figure 2. Effect of NVP-BEZ235 on the phosphorylation status ofPI3K/Akt/mTOR signaling components. A, cells were treated withincreasing concentrations of NVP-BEZ235 for 24 h, collected,lysed, and analyzed by Western blot. B, Western blot analysis ofcells treated with 200 nmol/L NVP-BEZ235 for 1, 3, 6 h.Representative of three different experiments (A and B).

2

Cancer Research



of flowslightapoptoall, thethe grboth aalso sutic indof nor

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cytometry of PI-stained samples, documented only aincrease in G0/G1 cells (Supplementary Fig. S1B). Nosis was detected in these cells (data not shown). Over-se findings showed that NVP-BEZ235 potently reducedowth of T-ALL cell lines and that this effect was due topoptosis and cell cycle arrest. Moreover, these resultsggested that the drug could have a favorable therapeu-ex, as it did not significantly affect the proliferationmal CD4+ T lymphocytes.

EZ235 induces autophagymTORC1 is an inhibitor of autophagy (31), it was in-ated whether NVP-BEZ235 could indeed induce auto-in T-ALL cells. Induction of autophagy was confirmednsmission electron microscopy analysis, the goldenard for autophagy studies, which documented thece of autophagic vacuoles in the cytoplasm of NVP-5–treated CEM-S cells. Similar results were detectedLT-4 cells (Supplementary Fig. S2A). In CEM-S cellsd with NVP-BEZ235 for 16 hours, we observed an in-in Beclin-1, a well-established autophagy marker

2; Supplementary Fig. S2B). In addition, we studiedeavage of LC3, which is the only known mammalianin that stably associates with the autophagosomeranes (33). Indeed, LC3 can be detected as LC3-Aolic) and LC3-B (membrane bound and enriched intophagic vacuole fraction) forms. NVP-BEZ235 treat-
caused an increase in LC3-B (cleaved fragment;ementary Fig. S2B). Because it is now emerging that
It wwith

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hagy could represent a mechanism of protectionmented by cells treated with mTORC1 inhibitorst was investigated if an autophagy inhibitor, chloro-, could potentiate the proapoptotic effects of NVP-5. However, chloroquine actually slightly decreasedmber of apoptotic CEM-S cells when used in combi-with NVP-BEZ235, even if this decrease was not

tically significant (Supplementary Fig. S2C). Theses implied that autophagy induced by NVP-BEZ235protective effect against apoptosis.

EZ235 affects PI3K/Akt/mTOR signaling in T-ALLnesstern blot analysis showed a dose-dependent decrease473 p-Akt after 24 hours of treatment with the drug incell lines (Fig. 2A). BE-13 cells did not display Akt

horylated on Ser473. Total Akt levels were unaffectedP-BEZ235. mTORC1 downstream substrates (p70S6K,1, and S6RP) were also efficiently dephosphorylatedP-BEZ235, whereas their total levels did not changeA). A time-dependent study was also performed, whichented that NVP-BEZ235 (200 nmol/L) dephosphory-kt and S6RP already after 1 hour of treatment, where-S6K and 4E-BP1 dephosphorylation was detected afterrs of treatment (Fig. 2B).

EZ235 synergizes with chemotherapeutic drugs

3. The combination of NVP-BEZ235 with conventional chemotherapeutic agents is synergistic in T-ALL cell lines. Cells were cultured in thee of cyclophosphamide (Cyclo; nmol/L) or Ara-C and NVP-BEZ235 (NVP; nmol/L) alone or in combination at a fixed ratio. The combined treatment

as investigated whether NVP-BEZ235 could synergizedrugs commonly used for treating T-ALL patients.




T-ALLalone,a fixeBEZ23ysis ofall thestrongstrongthat w8402(data

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and lechemwhethvincriscell linfor prbone mcells oNVP-Bing froCD45+

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cell lines were incubated for 24 hours with the drugsNVP-BEZ235 alone, or the drugs plus NVP-BEZ235 atd ratio (NVP-BEZ235/cyclophosphamide, 1:10; NVP-5/Ara-C, 1:1). MTT assays were then performed. Anal-the results proved that NVP-BEZ235 synergized withtested drugs; however, the synergism was particularlywith Ara-C. CI analysis documented the existence of asynergism (CI < 0.3) at NVP-BEZ235 concentrationsere well below its IC50 (Fig. 3). Moreover, in RPMI-cells, NVP-BEZ235 synergized with dexamethasonenot shown).

EZ235 reduces chemoresistance to vincristineed in Jurkat cells by coculturing with MS-5al cellsause interactions between bone marrow stromal cellsukemic cells are thought to be responsible for decreasedotherapeutic drug sensitivity (35), we investigateder NVP-BEZ235 could increase the cytotoxicity oftine in a coculture system. We used the murine stromale MS-5, which is known to provide long-term supportimitive hematopoietic progenitors and to mimic thearrow microenvironment (36). Coculture with stromalnly slightly decreased the sensitivity of Jurkat cells toEZ235, with the percentage of apoptotic cells decreas-m 15% to 11% (Fig. 4A). Flow cytometric analysis ofcells showed that coculture with stromal cells signifi-(P < 0.01) decreased the sensitivity to vincristine ofcells when compared with cells growing as suspensiones, as documented by the percentage of Annexin V-positive cells. However, NVP-BEZ235 restored vincris-nsitivity to the levels measured in suspension culturesd with vincristine alone. Nevertheless, the efficacy ofmbined treatment in the presence of stromal cells wasr to that detected in suspension cultures (Fig. 4A). Thisly reflects activation of additional survival pathways byal cells. To determine whether the effect of NVP-BEZ235kat cells was direct or indirect via an effect on MS-5 sig-or viability, we performed the following experiments.PI3K/Akt/mTORC1 signaling was studied by flow cyto-in MS-5 stromal cells cocultured with Jurkat cells, it wasle to document that NVP-BEZ235 did not affect thehorylation levels of both Ser473 p-Akt and Thr37/46 p-1 (Fig. 4B). Moreover, either vincristine or NVP-BEZ235did not affect in a statistically significant manner MS-5ability, whereas with the combined treatment, 85% ofal cells were still viable (Fig. 4C).

lymphoblasts are sensitive to NVP-BEZ235better assess the effectiveness of NVP-BEZ235 as atial therapeutic agent in T-ALL, we examined sevenric T-ALL patient samples isolated from the bonew or peripheral blood for the levels of PTEN, Ser473Thr37/46 p-4E-BP1, and Ser235/236 p-S6RP, as welltheir sensitivity to NVP-BEZ235, using flow cyto-

c and MTT assays. All of the patients displayed
ced phosphorylation of Ser473 p-Akt, Thr37/46 p-1, and Ser235/236 p-S6RP (not shown). As to PTEN,
of theefficac

r Res; 70(20) October 15, 2010


atient had very low levels whereas the six othersigher levels (Fig. 5A). These results were in agreementhe gene expression profiles (not shown). T-ALL lym-ast samples were treated with increasing concentra-of NVP-BEZ235, and cell survival was analyzed byssays. A marked reduction of cell viability at 96 hoursetected. The IC50 for patient samples ranged betweend 50 nmol/L NVP-BEZ235 (Fig. 5B). Flow cytometricis documented a decrease in the levels of p-4E-BP1-S6RP in NVP-BEZ235–treated samples (Fig. 5C).stingly, rapamycin treatment did not result in dephos-lation of 4E-BP1, although it efficiently dephosphory-S6RP. These results were confirmed by Western blotis (Fig. 5C and D). Overall, these findings show thatEZ235 has a potent cytotoxic activity also againstry cells from T-ALL patients with upregulated PI3K/TORC1 signaling.

EZ235 targets the SP of T-ALL cell lines andt lymphoblasts

ally, we sought to determine whether NVP-BEZ235target the T-ALL SP. This subpopulation, which over-ses ABCG2 (also referred to as breast cancer resis-protein or BRCP) and other ABC plasma membraneorters, is thought to share some properties of cancercells (CSC; ref. 37). This has been shown in solids (38, 39) and acute myelogenous leukemia (40). How-candidate CSCs have been successfully identified inof a murine model of adult T-cell leukemia/lympho-

1). This suggests that the SP of T-ALL shares somerties with CSCs. The SP of T-ALL cells was identifiedlive-cell DNA-binding dye Hoechst 33342. As a con-oechst 33342 staining could be inhibited after incuba-ith either of the high-specificity ABCG2 transportertors fumitremorgin C or KO143. A decrease in thent of SP cells was evident in samples treated withNVP-BEZ235 or rapamycin for 24 hours (Fig. 6A) in-4, CEM-S, and BE-13 cells. Next, we analyzed by flowetry the plasma membrane levels of the most impor-BC transporters in the SP of CEM-S cells, which dis-no expression of both P-gp and MRP1 but high levels

CG2 (Fig. 6B).hNVP-BEZ235 and rapamycin targeted the SP of lympho-from T-ALL patients (two representative patients are). Expression of ABCG2 on SP cells was documented bystainingwithHoechst 33342 and an antibody that recog-n extracellular epitope of the transporter (Fig. 6C and D).

ssion

PI3K/Akt/mTOR pathway is a recently identified po-l target for therapeutic intervention in T-ALL. Becauseomplexity and extensive crosstalk with other signalingdes, therapeutic targeting of the PI3K/Akt/mTORrk at multiple molecular levels may provide better anti-effects than selective inhibition of only one component
pathway. In fact, one potential reason for the limitedy of single inhibitors in this pathway is the presence of
Cancer Research



Figuremarrow(VCR; 1cytomeJurkatgating(10 nmoversus


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4. NVP-BEZ235 reduces chemoresistance to vincristine induced in Jurkat cells by coculturing with MS-5 stromal cells, which mimic the bonemicroenvironment. A, Jurkat cells, growing either as suspension cultures or in a coculture system with MS-5 cells, were treated with vincristine0 nmol/L) or NVP-BEZ235 (NVP; 200 nmol/L), alone or in combination, and then analyzed by Annexin V-FITC/CD45-PE staining using flowtry. The percentages of healthy and apoptotic cells are indicated in top left and top right quadrants, respectively. B, MS-5 cells cocultured withcells were stained with AlexaFluor 488–conjugated anti–p-Akt or anti–p-4E-BP1. Jurkat cells were excluded from the analysis by electronicallyon CD45-PE+ cells. CTRL, untreated cells. NVP, cells treated for 24 h with 200 nmol/L NVP-BEZ235. C, MS-5 cells were incubated with vincristine

+
l/L) or NVP-BEZ235 (200 nmol/L), alone or in combination, and then the percentage of Annexin-V cells was analyzed by flow cytometry. *, P < 0.05,untreated cells (CTRL). Representative of three experiments (A–C).
Cancer Res; 70(20) October 15, 2010acrjournals.org 8103

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ng feedback loops operating through p70S6K and PI3Kxposure to a dual catalytic PI3K/mTOR inhibitor mightre be sufficient to avoid PI3K/Akt pathway reactivation
oreover, targeting this pathway at multiple levels mayt the development of drug resistance (42). In our previ-
on T-patien

r Res; 70(20) October 15, 2010


rk, we have documented the efficacy of dual PI3K/mTORion in T-ALL cells using PI-103 (20). Here, we showed they of the novel dual PI3K/mTOR inhibitor NVP-BEZ235

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ALL cell lines and lymphoblats. NVP-BEZ235 is an ATP c

20. © 2010 American Assoc

5. NVP-BEZ235 cytotoxicityT-ALL primary cellsing constitutive activationI3K/Akt/mTOR pathway.grams showing PTEN statusrepresentative patient (pt)s. B, MTT assay of T-ALLreated with NVP-BEZ235 foroints, mean of at least twot experiments; bars, SD.presentative patients areC, patient samples werewith 100 nmol/L rapamycinnmol/L NVP-BEZ235 ford then analyzed by flowtry for p-S6RP andP1. D, Western blot analysisrepresentative patients treated with rapamycin00 nmol/L) or NVP-BEZ23500 nmol/L) for 48 h, thenollected, and probed withies against p-4E-BP1ctin. Representative offerent experiments. CTRL,

sts derived from T-ALLompetitor that potently

Cancer Research

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s the kinase activity of both p110 PI3K and mTORC1/2,efficacy in advanced solid tumors is currently being eval-in phase 1 and 2 clinical trials (22). A comparisonen PI-103 and NVP-BEZ235 documented that the latterund was significantly more powerful toward T-ALL cellsused on an equimolar basis. NVP-BEZ235 inducedsis, which led to a cleavage of both procaspase-8 andpase-9, suggesting that both the intrinsic and extrinsicays of apoptosis are activated on exposure to the drug.f downregulation of PI3K/Akt/mTOR signaling is usuallyiated with activation of the intrinsic pathway (43),e-8 activation by the dual PI3K/mTOR inhibitor PI-103en reported in leukemic cells (44). In breast cancer cells,EZ235 activated caspase-2, which played an importantthe apoptotic process (45).
reover, treatment with NVP-BEZ235 resulted in aaccumulation in the G1 phase of the cell cycle and
tant magents

ents. CTRL, cells stained with Hoechst 33342 only. Representative of two sepant antibody); white histograms, anti-ABCG2 antibody.

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sphorylated pRb on Ser807/811, which are two of thecritical residues for pRb activity in cell cycle progres-46). Consistent with its inhibitory action on bothC1 and mTORC2, NVP-BEZ235 dephosphorylatedK/4E-BP1/S6RP as well as Akt on Ser473 in T-ALL cells.ence indicates that the modulation of autophagy is antant component of tumorigenesis, making it a possibleeutic target. The PI3K/Akt/mTOR pathway is impor-n inhibiting autophagy (47). We observed that auto-is activated in response to NVP-BEZ235 treatment incell lines. This observation could be very important,e recent studies have documented that tumorigenesislated by consitutively activated PI3K/Akt signaling isto the ability to inhibit autophagy but not apoptosis,the possibility that autophagy may also be an impor-
echanism underlying the response to therapeutictargeting the PI3K/Akt/mTOR pathway (47).
6. NVP-BEZ235 and rapamycin target the SP of T-ALL cells. A, T-ALL cell lines treated with rapamycin (rapa) or NVP-BEZ235 (NVP) for 24 hained with Hoechst 33342 dye (5 μg/mL) in the presence or absence of KO143 (0.1 μmol/L) and then analyzed by flow cytometry. The SP, whichared in the presence of KO143, was gated and shown as a percentage of the whole viable cell population. B, flow cytometric analysis of CEM-S SPained with PE-conjugated antibodies to ABCG2, P-gp, and MRP1. Black histograms, negative control (irrelevant antibody); white histograms,C transporter antibody. C and D, flow cytometric analysis of the SP and ABCG2 in two representative patient samples. T-ALL primary cells wereed for 24 h with rapamycin (100 nmol/L) or NVP-BEZ235 (200 nmol/L). Fumitremorgin C was used at 10 μmol/L. Representative of two separate

rate experiments. D, black histograms, negative control




Beccells orole inNVP-Bsugges(NVP-marrostronglymphized bover,S6RPdid noportedtargetthan rThe

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ause survival signals generated by cytokines secreted byf the bone marrowmicroenvironment play an importantthe emergence of drug resistance, we asked whetherEZ235 could diminish these survival signals. Our resultst a potential clinical activity of a combination therapyBEZ235 plus vincristine) even in the presence of bonew stromal cells. Remarkably, NVP-BEZ235 displayed a(in the low nanomolar range) cytotoxic activity againstoblasts from patients with pediatric T-ALL character-y enhanced levels of p-Akt, p-4E-BP1, and p-S6RP. More-it caused the dephosphorylation of both 4E-BP1 andin these samples, whereas treatment with rapamycint induce 4E-BP1 dephosphorylation. Therefore, as re-for acute myelogenous leukemia cells (16), agents thatmTORC1 activities globally could be more beneficialapamycin/rapalogues for T-ALL patients (16).difficulty in eradicating tumors might result from thentional treatments targeting the bulk of the tumorut not the CSCs. Therefore, strategies that eliminatecells could have significant clinical implications. Wehown that both NVP-BEZ235 and rapamycin markedlyd the T-ALL SP, which might correspond, at least ino CSCs. It is still unclear whether NVP-BEZ235 wasly cytotoxic to the SP cells or it simply blocked ABCG2y (and thus Hoechst dye extrusion), as it has beenusly documented that membrane localization of ABCG2use bone marrow cells was dependent on Akt activityowever, NVP-BEZ235 targeted the SP of BE-13 cells
o not display Akt activation. Moreover, rapamycin alsoly reduced the SP of T-ALL, and our previous results
ReceOnlineF

tierrez A, Sanda T, Grebliunaite R, et al. High frequency of PTEN,K, AKT abnormalities in T-cell acute lymphoblastic leukemia.od 2009;114:647–50.

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ted that rapamycin was not as effective as PI-103 inng p-Akt levels in T-ALL cells (20).se observations suggest that NVP-BEZ235 could bely cytotoxic to the SP cells due to inhibition of mTORC1y. Indeed, the key role played by mTORC1 signaling inrvival of leukemic stem cells in mouse models of acuteoblastic leukemia is now beginning to emerge (49, 50).ver, additional experiments are required to furthers this issue.en together, our preclinical findings strongly suggestVP-BEZ235, either alone or in combination with tradi-chemotherapeutic drugs, could be a valuable com-in the treatment of those T-ALL patients displaying

rognosis.

otential conflicts of interest were disclosed.

Support

azione del Monte di Bologna e Ravenna, MinSan 2008 “Molecularin pediatric sarcomas and leukemias against IGF-1 receptor system,”IN 2008 (A.M. Martelli).costs of publication of this article were defrayed in part by the paymentcharges. This article must therefore be hereby marked advertisement innce with 18 U.S.C. Section 1734 solely to indicate this fact.

ived 05/21/2010; revised 07/30/2010; accepted 08/13/2010; publishedirst 09/28/2010.

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2010;70:8097-8107. Published OnlineFirst September 28, 2010.Cancer Res Francesca Chiarini, Cecilia Grimaldi, Francesca Ricci, et al. NVP-BEZ235 against T-Cell Acute Lymphoblastic Leukemia3-Kinase/Mammalian Target of Rapamycin Inhibitor Activity of the Novel Dual Phosphatidylinositol

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