Experimental Therapeutics, Molecular Targets, and Chemical Biology

The Novel Tubulin-Targeting Agent Pyrrolo-1,5-Benzoxazepine-15Induces Apoptosis in Poor Prognostic Subgroups of ChronicLymphocytic Leukemia

Anthony M. McElligott,1 Elaina N. Maginn,1 Lisa M. Greene,2 Siobhan McGuckin,3

Amjad Hayat,1,6 Paul V. Browne,3 Stefania Butini,4 Giuseppe Campiani,4

Mark A. Catherwood,5 Elisabeth Vandenberghe,3 D. Clive Williams,2

Daniela M. Zisterer,2 and Mark Lawler1

1John Durkan Research Laboratories, Institute of Molecular Medicine, and 2School of Biochemistry and Immunology, Trinity CollegeDublin, Dublin, Ireland; 3Department of Haematology, St. James's Hospital, Dublin, Ireland; 4European Research Centre for Drug Discoveryand Development, University of Siena, Siena, Italy; 5Department of Haematology, Belfast City Hospital, Belfast, Northern Ireland; and6Department of Haematology, University College Hospital, Galway, Ireland

AbstractPyrrolo-1,5-benzoxazepine-15 (PBOX-15) is a novel microtu-bule depolymerization agent that induces cell cycle arrestand subsequent apoptosis in a number of cancer cell lines.Chronic lymphocytic leukemia (CLL) is characterized byclonal expansion of predominately nonproliferating matureB cells. Here, we present data suggesting PBOX-15 is a poten-tial therapeutic agent for CLL. We show activity of PBOX-15 insamples taken from a cohort of CLL patients (n = 55) repre-senting both high-risk and low-risk disease. PBOX-15 exhib-ited cytotoxicity in CLL cells (n = 19) in a dose-dependentmanner, with mean IC50 of 0.55 μmol/L. PBOX-15 significantlyinduced apoptosis in CLL cells (n = 46) including cells withpoor prognostic markers: unmutated IgVH genes, CD38 andzeta-associated protein 70 (ZAP-70) expression, and fludara-bine-resistant cells with chromosomal deletions in 17p. Inaddition, PBOX-15 was more potent than fludarabine in in-ducing apoptosis in fludarabine-sensitive cells. Pharmaco-logic inhibition and small interfering RNA knockdown ofcaspase-8 significantly inhibited PBOX-15–induced apoptosis.Pharmacologic inhibition of c-jun NH2-terminal kinase inhib-ited PBOX-15–induced apoptosis in mutated IgVH and ZAP-70

−

CLL cells but not in unmutated IgVH and ZAP-70+ cells. PBOX-

15 exhibited selective cytotoxicity in CLL cells compared withnormal hematopoietic cells. Our data suggest that PBOX-15 re-presents a novel class of agents that are toxic toward bothhigh-risk and low-risk CLL cells. The need for novel treatmentsis acute in CLL, especially for the subgroup of patients withpoor clinical outcome and drug-resistant disease. This studyidentifies a novel agent with significant clinical potential.[Cancer Res 2009;69(21):8366–75]

IntroductionB-Chronic lymphocytic leukemia (CLL) is a common leukemia

characterized by the accumulation of long-lived CD5-positivemonoclonal B cells in the bone marrow, peripheral lymphoid

organs, and blood (1). It is believed that CLL is primarily due todefects in apoptotic mechanisms in malignant cells rather than de-fects in control of lymphoproliferation, although this is the subjectof some reevaluation (2). The clinical course of CLL is heteroge-neous, with some patients displaying stable disease, which oftenrequires no treatment other than “watchful waiting,” whereas otherpatients have aggressive disease necessitating early intervention.Several poor prognostic markers have been identified, includingpresence of unmutated immunoglobulin heavy chain genes (U-IgVH), cell surface CD38 expression, cytoplasmic zeta-associatedprotein 70 (ZAP-70) expression, and chromosomal abnormalitiessuch as deletion in 17p (del17p) and del11q23. In contrast, favorableoutcome is associated with presence of del13q and mutated IgVH(M-IgVH). Current treatment paradigms for CLL involve use of alky-lating agents, such as fludarabine, and monoclonal antibodies(mAb; ref. 3).Pyrrolo-1,5-benzoxazepines (PBOX) are a family of novel com-

pounds, which we have previously shown to induce cell cycle arrestand apoptosis in leukemia cell lines (4, 5) and inhibit tumor growthin an in vivo breast carcinoma model (6). More recently, we haveshown that two of the more active members of the family, pyrrolo-1,5-benzoxazepine-6 (PBOX-6) and pyrrolo-1,5-benzoxazepine-15(PBOX-15), cause depolymerization of microtubules and disassem-bly of tubulin in vitro (7).Microtubules, key components of the cytoskeleton, are ex-

tremely important in mitosis and cell division and are thereforean important target for anticancer drugs. A chemically diversegroup of antimitotic drugs target microtubules and have beenused with great success in the treatment of cancer (8). These canbe broadly classified either as tubulin polymerizers (e.g., taxanes) ordepolymerizers (the Vinca alkaloids, colchicines, and nocodazole).Proliferating cancer cells treated with microtubule targeting agents(MTA) arrest in the G2-M phase of the cell cycle and eventuallyundergo apoptotic cell death. Several well-characterized MTAshave been shown to induce apoptosis in CLL cells, including colchi-cine (9, 10), vincristine (11), and nocodazole (12), as well as novelagents with microtubule targeting activity (13, 14). There seems tobe a specific effect of microtubule depolymerization agents in CLL,as Taxol has been reported not to induce apoptosis in CLL cells(12). The mechanism by which MTAs induce apoptosis in CLL isunclear. As circulating CLL cells are noncycling, the mechanismof action cannot be accounted for by induction of mitotic arrest.However, microtubules have been shown to be involved in B-cellreceptor signaling via interaction of tubulin with the tyrosine kinase

Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).

Requests for reprints: Tony McElligott, Institute of Molecular Medicine, TrinityCentre, St. James's Hospital, Dublin 8, Ireland. Phone: 353-1-896-3276; Fax: 353-1-410-3476. E-mail: tony.mcelligott@tcd.ie.

©2009 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-09-0131

8366Cancer Res 2009; 69: (21). November 1, 2009 www.aacrjournals.org

Research. on May 8, 2016. © 2009 American Association for Cancercancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 13, 2009; DOI: 10.1158/0008-5472.CAN-09-0131

Syk and downstream signaling molecules Cbl and Vav (15). B-cellreceptor signaling differs between the IgVH subgroups of CLL cells,with U-IgVH cells exhibiting signal transduction following cross-linking of B-cell receptor, whereas cells with M-IgVH are unrespon-sive (16). This difference in B-cell receptor signaling may be due toZAP-70, a critical T-cell receptor–associated tyrosine kinase (17),which is associated with U-IgVH (18).In this study, we show that PBOX-15 potently and selectively

induces apoptosis in CLL cells. We show that PBOX-15 is equi-potent in poor prognostic subgroups of CLL (CD38+, ZAP-70+,and U-IgVH genes), including fludarabine-resistant cells withdel17p. We investigated the mechanism of action and showthat PBOX-15–induced apoptosis occurs via a caspase-8–dependent mechanism. In addition, we suggest a role for c-junNH2-terminal kinase (JNK) activation in PBOX-15–inducedapoptosis in cells with favorable prognostic indicators (M-IgVHand ZAP-70−).

Materials and MethodsPBOX-15. PBOX-15 [pyrrolobenzoxazepine 4-acetoxy-5-(1-naphthyl)

naphtha pyrrolo[1,4]-oxazepine; Fig. 1A] was synthesized as described pre-viously (19, 20).

Patient samples and cell lines. Fifty-five patients referred to St.James's Hospital, Dublin were recruited to this study. All patients exhibitedmorphologic and immunophenotypic criteria of CLL (CD5/19+, CD23+,FMC7−, weak surface immunoglobulin, and light chain restriction), hadstage A disease, and had not undergone previous treatment. Written in-formed consent was obtained before blood collection. In a concurrentstudy (21), we analyzed IgVH mutational status, karyotype, and CD38and ZAP-70 expression in a subset of these patients (Table 1). Bloodand bone marrow were also obtained from healthy donors. The studywas approved by the St. James's Hospital and Adelaide and Meath incor-porating the National Children's Hospital Ethics Committee. Peripheralblood mononuclear cells (PBMC) were isolated from herparinized periph-eral blood by Lymphoprep (Axis-Shield) density gradient centrifugationand incubated in RPMI 1640 with Glutamax supplemented with 10%FCS, 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen;complete medium). EBV-transformed CLL cell lines, PGA1 (M-IgVH) andHG3 (U-IgVH), were obtained from Prof. Anders Rosén (Linköping Univer-sity, Linköping, Sweden; ref. 23). Cell lines were maintained in completemedia without antibiotics.Granulocyte-macrophage colony-forming unit assay of bone mar-

row progenitor cells. The colony-forming potential of normal donorbone marrow progenitor cells was assessed using granulocyte-macro-phage colony-forming unit (CFU-GM) assays. Bone marrow mononuclearcells were isolated as described above. Mononuclear cells (1 × 106) wereincubated with PBOX-15 in 1 mL of complete medium. After incubation,a 200-μL aliquot was removed and added to 1.8 mL of CFU-GM Metho‐Cult methylcellulose medium (StemCell Technologies) and plated in trip-licate in 2-cm2 cell culture dishes. Colonies with ≥30 cells were countedafter 14 d.Viability and apoptosis assays. An MTT assay (Roche) was used to

quantify cell viability. PBMCs (1 × 106) were seeded in 96-well plates, andMTT assay was carried out according to the manufacturer's protocol. IC50values were calculated using GraphPad Prism version 4.Apoptosis was quantified by Annexin V binding assays as described

previously (24). PBMCs (5 × 106) or PGA1 and HG3 cells (5 × 105) weretreated with PBOX-15, 2-fluoroadenine-9-β-D-arabinofuranoside (fludara-bine), or nocodazole (Sigma-Aldrich) in 1 mL of complete medium in24-well plates. Caspase and JNK inhibitors (Calbiochem) were added1 h before treatment. At the appropriate time point, cells were stainedwith Annexin V-FITC (IQ Products) and propidium iodide (PI; MolecularProbes) before collection of data via flow cytometry and analysis byCellQuest software (FACSCalibur, BD Biosciences). Apoptosis in normaldonor B cells was assessed by incubation of PBMCs with phycoerythrin-labeled CD19 antibody (BD Biosciences) and subsequent analysis ofAnnexin V binding in CD19+ B cells. Mitochondrial integrity was as-sessed by incubating cells with the membrane potential–sensitive dyerhodamine-123 (Molecular Probes; 50 ng/mL in complete medium) for30 min and analysis by flow cytometry.Caspase-8 knockdown. PGA1 cells (4 × 105) were incubated with 1

μmol/L Accell SMARTpool caspase-8 small interfering RNA (siRNA) orAccell nontargeting siRNA pool (Dharmacon) in 1 mL Accell siRNA deliverymedium containing 3% FCS in 24-well plates. Following incubation for 72 h,PBOX-15 was added and cells were incubated for a further 16 h before anal-ysis of caspase-8 expression by immunoblotting and of apoptosis inductionby Annexin V/PI staining.Protein analysis. Immunoblotting of whole-cell lysates was done as pre-

viously described (25). Proteins were resolved by SDS-PAGE and transferredonto polyvinylidene difluoride membrane (Bio-Rad). After probing for Bcl-2(mouse mAb; Calbiochem), caspase-8 (mouse mAb; Cell Signaling), or phos-pho-JNK (rabbit mAb; Cell Signaling), blots were incubated with the appro-priate horseradish peroxidase–conjugated secondary antibody. Allmembranes were reprobedwith anti-actin antibody to confirm equal loading.Staining of microtubules. Following treatment, cells were cytospun,

air-dried, and fixed in 100% methanol. Fixed cells were stained withanti–α-tubulin mouse mAb (clone DM1A), FITC-conjugated goat anti-mouse secondary antibody (Sigma-Aldrich), and Hoechst 33258 (MolecularProbes) as described previously (26). Projected images from a z-series of

Figure 1. PBOX-15 is cytotoxic to primary CLL cells and disrupts microtubules.A, PBOX-15. B, CLL cells isolated from patient #22 were assessed forcell survival by MTT assay following treatment for 24, 48, and 72 h with0.003 to 10 μmol/L of PBOX-15. Bars, SD of triplicate samples. C, CLL cellsisolated from 19 patients were incubated with PBOX-15 for 24 h and IC50

values were calculated from MTT assays. D, confocal microscopy of CLL cells(patient #10) stained for microtubules with anti–α-tubulin antibody followingtreatment for 24 h with 0.1% (v/v) ethanol (control) or 1 μmol/L PBOX-15.Arrow, microtubules radiating from centrosome. Bar, 10 μm. Representative ofthree independent experiments.

PBOX-15 Induces Apoptosis in CLL Cells

8367 Cancer Res 2009; 69: (21). November 1, 2009www.aacrjournals.org

Research. on May 8, 2016. © 2009 American Association for Cancercancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 13, 2009; DOI: 10.1158/0008-5472.CAN-09-0131

Table 1. Clinical and biological characteristics of CLL patients

Patient # Age Sex Karyotype CD38* IgVH† ZAP-70‡ IC50 (μmol/L) AV/PI Cas Inh JNK Inh Flu

1 83 M del13q − M − 0.62 ✓

2 62 M normal − U − ✓ ✓

3 52 M normal + U − ✓ ✓

4 71 M del17p + nd nd ✓ ✓

5 72 M del13q − M − ✓

6 48 M nd nd nd nd ✓ ✓

7 55 M del13q − M − ✓ ✓

8 77 F normal − M − 0.189 76 M del13q − M − 0.4710 79 F del13q + M − 0.43 ✓

11 61 M del13q − M − ✓ ✓ ✓

12 76 F nd − M − ✓ ✓ ✓

13 63 M normal − U nd 0.49 ✓

14 57 M normal − nd nd ✓ ✓

15 62 M normal + nd nd ✓ ✓ ✓

16 50 M del11q − U − ✓

17 39 F del13q − M − ✓ ✓ ✓

18 55 M normal − nd nd ✓

19 64 M del13q + M + 0.60 ✓ ✓ ✓

20 62 F del13q − nd nd ✓ ✓ ✓

21 81 F nd − M nd ✓

22 72 F del13q − M − 0.3723 72 F normal − M − 0.5424 50 F normal − M − 0.54 ✓

25 74 F del17p,del13q14 + M − ✓ ✓

26 57 F normal − nd nd ✓ ✓

27 84 F normal − M − 0.4128 64 F del13q − nd nd ✓

29 60 F trisomy12 + U + 0.77 ✓

30 75 F trisomy12 + M nd ✓ ✓

31 66 M normal − nd nd ✓ ✓

32 82 F normal − M − ✓ ✓ ✓

33 82 F del13q − U + 1.1 ✓ ✓ ✓

34 84 M del13q − U + 0.84 ✓ ✓ ✓

35 60 M del13q − U + ✓ ✓ ✓

36 57 M nd nd nd nd ✓

37 61 M normal − nd nd ✓ ✓ ✓

38 69 M del13q − U − ✓ ✓ ✓ ✓

39 62 M del13q − M − ✓ ✓ ✓

40 64 M normal − nd nd ✓ ✓

41 54 F nd + U + ✓ ✓ ✓

42 82 F del13q − M − 0.3943 67 M trisomy12 + U − 0.4644 54 F trisomy12 + M − 0.61 ✓

45 69 M del13q − M − ✓ ✓ ✓ ✓

46 56 M normal − M − ✓ ✓ ✓

47 79 M del13q + U + ✓ ✓

48 75 M nd nd nd nd ✓ ✓ ✓

49 64 M normal + M − 0.84 ✓

50 64 F nd nd nd nd ✓ ✓ ✓

51 85 F normal − M − 0.3352 75 M nd nd nd nd 0.5553 59 F del13q − M − ✓ ✓ ✓

54 51 F del17p + U + ✓ ✓

55 70 M del13q − U − ✓ ✓ ✓

NOTE: IC50 values for PBOX-15 indicated.✓ indicates samples used for PBOX-15–induced apoptosis assay (AV/PI), caspase inhibitor assays (Cas Inh),JNK inhibitor assays (JNK Inh), and fludarabine-induced apoptosis assay (Flu). nd, not determined.*Positive patients had ≥30% leukemic cells positive for CD38 (21).†IgVH gene mutational status: M, mutated; U, unmutated.‡Positive patients had ≥20% leukemic cells positive for ZAP-70 (22).

Cancer Research

8368Cancer Res 2009; 69: (21). November 1, 2009 www.aacrjournals.org

Research. on May 8, 2016. © 2009 American Association for Cancercancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 13, 2009; DOI: 10.1158/0008-5472.CAN-09-0131

images were captured using Olympus 1X81 microscope coupled with Fluo-view Ver 1.5 software (Olympus).Statistical analysis. Differences in apoptosis between treated and un-

treated cells were analyzed by Student's t test. Association between prog-nostic markers and apoptosis was analyzed by Mann-Whitney U test andFisher's exact test. Correlations were determined by Pearson's method. Forall tests, P < 0.05 was considered significant.

Results

PBOX-15 is cytotoxic to CLL cells and disrupts CLL cellmicrotubules. Initially, we investigated the cytotoxic effects ofPBOX-15 using an MTT assay. As CLL cells are growth arrestedin G0-G1 (data not shown), this assay quantifies cell viability andnot proliferation. Cell viability was determined after treatmentwith a range of PBOX-15 concentrations (3 nmol/L–10 μmol/L)for up to 72 hours (Fig. 1B), and a sigmoidal dose responseallowed cytotoxicity to be expressed as an IC50 value. In thistypical result, maximal PBOX-15–induced cytotoxicity was in-duced after 48 hours; however, there was no significant increasein IC50 values after 24 hours. In 19 patient samples analyzed,

IC50 values ranged between 0.18 and 1.1 μmol/L (mean,0.55 μmol/L; Fig. 1C). We have previously shown that PBOX-15targets tubulin polymerization in cancer cell lines and T lym-phoctyes (7, 27). To determine whether PBOX-15 exerts a similareffect on CLL cells, the microtubule architecture was analyzed.Control cells exhibited an organized microtubule network radi-ating from the centrosome. However, following treatment with1 μmol/L PBOX-15 for 24 hours, this microtubule organizationwas completely disrupted with markedly decreased tubulinstaining (Fig. 1D).PBOX-15 induces apoptosis in CLL cells. Next, we investigat-

ed the type of cell death mediated by PBOX-15. CLL cells isolatedfrom 11 patients were treated with 0.1 to 10 μmol/L of PBOX-15for 24 and 48 hours, and a dose response induction of apoptosiswas observed (Fig. 2A). Significant induction of apoptosis was in-duced in cells treated with 1 μmol/L PBOX-15 for 24 hours, withno further significant increase observed following treatment for 48hours. Analysis of 46 patients' samples showed that 1 μmol/LPBOX-15 significantly induced apoptosis in CLL cells (Fig. 2B). Al-though there was a wide range in both spontaneous (3–43%) andPBOX-15–induced apoptosis (22–90%), apoptosis increased in

Figure 2. PBOX-15 induces apoptosis in CLL cells. A, CLL cells isolated from 11 patients were treated for 24 and 48 h with the indicated concentrations of PBOX-15.Apoptosis was measured by Annexin V/PI assay with apoptotic cells determined by Annexin V–positive staining. B, analysis of 46 primary CLL samples showedsignificant induction of apoptosis following 24 h incubation with 1 μmol/L PBOX-15 compared with 0.1% ethanol control (P < 0.0001). C, PBOX-15 induced apoptosis incells with (i) M-IgVH (n = 19) and U-IgVH (n = 13), (ii) CD38+ (n = 13) and CD38− (n = 29) cells, (iii) ZAP-70+ (n = 8) and ZAP-70− (n = 21) cells, and (iv) indicatedkaryotypes: del13q (n = 18), del17p (n = 3), normal (n = 14), and trisomy 12 (n = 3). D, induction of apoptosis in PGA1 (M-IgVH) and HG3 (U-IgVH) cell lines followingtreatment with the indicated concentrations of PBOX-15 for 24 and 48 h. n = 3. Columns, mean; bars, SE.

PBOX-15 Induces Apoptosis in CLL Cells

8369 Cancer Res 2009; 69: (21). November 1, 2009www.aacrjournals.org

Research. on May 8, 2016. © 2009 American Association for Cancercancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 13, 2009; DOI: 10.1158/0008-5472.CAN-09-0131

every CLL sample following treatment (range of increase, 9–79%;median increase, 48%). Importantly, PBOX-15 induced similar le-vels of apoptosis in cells with markers for poor clinical outcome:U-IgVH (Fig. 2Ci), CD38

+ (Fig. 2Cii), ZAP-70+ (Fig. 2Ciii), and del17p(Fig. 2Civ). There was no correlation between spontaneous orPBOX-15–induced apoptosis and karyotypic status, IgVH muta-tional status, or CD38 or ZAP-70 expression. PBOX-15 induced ap-optosis in two CLL cell lines: PGA1 (M-IgVH) and HG3 (U-IgVH;Fig. 2D).PBOX-15 has reduced toxicity in normal hematopoietic

cells.We investigated the effects of PBOX-15 on normal bone mar-row donor progenitor cells using a CFU-GM assay. CFU-GM assaysare physiologically relevant toxicology models that have a high pre-dictive ability to detect myelosuppression due to chemotherapeu-tic agents (28). The effects of exposure of bone marrow progenitorcells isolated from three normal donors to 1 and 5 μmol/L PBOX-15 for up to 72 hours are shown in Fig. 3Ai–iii. There was a widerange of colony-forming ability between donor bone marrow pro-

genitor cells following treatment, reflecting the heterogeneity ofthe donors. Treatment with 1 μmol/L PBOX-15 for 24 hours re-sulted in no significant decrease in colony-forming ability in anyof the three donor samples. Further analysis of another five normalbone marrow donor samples showed that, whereas colony-formingability was reduced in four of eight samples following 1 μmol/LPBOX-15 treatment for 24 hours, the overall colony-forming abilitywas not significantly reduced following treatment (Fig. 3Aiv). Thisrelatively low cytotoxicity in normal bone marrow progenitor cellsmay be due to these cells recovering from PBOX-15–induced cellcycle arrest, resulting in no sustained effect on proliferative capac-ity. However, these results suggest that prolonged exposure toPBOX-15 may have significant cytotoxic effects on bone marrowprogenitor cells. Further studies, including the use of animal mod-els, are required before the bone marrow toxicity of PBOX-15 canbe fully assessed. We also investigated whether PBOX-15 exhibitedselective apoptosis induction in CLL cells compared with normal Bcells. No significant increase in apoptosis was detected in B cells

Figure 3. Effect of PBOX-15 on normalhematopoietic cells. A, effects of PBOX-15on the colony-forming ability of normaldonor bone marrow progenitor cells.The colony-forming ability of the progenitorcells was assayed in triplicate byCFU-GM. Columns, mean; bars, SE. i to iii,bone marrow progenitor cells wereincubated with the indicated concentrationsof PBOX-15 and 0.5% ethanol (control) for24, 48, and 72 h. iv, CFU-GM assay onbone marrow progenitor cells from eightdonors treated with 1 μmol/L PBOX-15 for24 h. Overall, there was no significantdifference between control andPBOX-15–treated cells (P = 0.16). B, effectof PBOX-15 on normal B cells. NormalPBMC (n = 4) were treated with 1% ethanol(control) or with 1 or 10 μmol/L PBOX-15for the indicated time points. Apoptosiswas assessed by Annexin V binding ofCD19+ B cells. Columns, mean apoptosis;bars, SE.

Cancer Research

8370Cancer Res 2009; 69: (21). November 1, 2009 www.aacrjournals.org

Research. on May 8, 2016. © 2009 American Association for Cancercancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 13, 2009; DOI: 10.1158/0008-5472.CAN-09-0131

following treatment with 1 μmol/L PBOX-15 for up to 72 hours(Fig. 3B).PBOX-15 is more potent than fludarabine in inducing apo-

ptosis in CLL cells. As the purine analogue fludarabine is a front-line agent in CLL therapy, we compared the sensitivity of CLL cellsto PBOX-15 and fludarabine. Fludarabine-induced apoptosis wasassessed using 2-fluoroadenine-9-β-D-arabinofuranoside, the active

nucleoside form of fludarabine. A typical result comparing theeffects of PBOX-15 and fludarabine shows that 1 μmol/L PBOX-15is more potent than the therapeutically relevant concentration of20 μmol/L fludarabine (29) in inducing apoptosis up to 72 hours(Fig. 4A). PBOX-15 at 1 μmol/L was significantly more potent ininducing apoptosis after 24 hours compared with 50 μmol/L fludar-abine (P < 0.02, n = 14; Fig. 4B). Del17p results in loss of expression ofp53 and is associated with short survival time and resistance to pu-rine analogues, such as fludarabine (30, 31). The three samples withdel17p were assessed for fludarabine- and PBOX-15–induced apo-ptosis after 24 hour treatment (Fig. 4C). Del17p cells exhibited com-plete resistance to spontaneous and fludarabine-induced apoptosis(levels of apoptosis <10%). However, del17p cells were sensitive toPBOX-15–induced apoptosis with levels of apoptosis ranging from25% to 63%.Mechanism of PBOX-15–induced apoptosis in CLL cells in-

volves activation of caspase-8. To investigate the mechanism ofPBOX-15–induced apoptosis in CLL cells, we investigated the effectof PBOX-15 on mitochondrial membrane depolarization, which isindicative of activation of the intrinsic pathway of apoptosis. Weshow mitochondrial membrane depolarization at 24 hours, but notat 2 hours, after treatment with PBOX-15 (Supplementary Fig. S1),suggesting that activation of this apoptotic pathway is not an earlyevent in PBOX-15–induced apoptosis.We have previously shown that proapoptotic PBOX com-

pounds result in phosphorylation and inactivation of the anti‐apoptotic protein Bcl-2 in a number of cancer cell lines (26, 32).Bcl-2 is phosphorylated in unstressed cells undergoing mitosis;however, hyperphosphorylation of Bcl-2 occurs in response to treat-ment with MTAs (33), including nonproliferating CLL cells (12, 34).Accordingly, we investigated phosphorylation and expression levelsof Bcl-2 in CLL cells following treatment with PBOX-15 (Fig. 5A). Nochange in protein levels of Bcl-2 was detected in any of four patientsamples following 24 hours of treatment with PBOX-15. Treatmentof the chronic myelogenous leukemia cell line K562 with PBOX-15resulted in extensive phosphorylation of Bcl-2; however, no phos-phorylation was detected in PBOX-15–treated CLL samples, sug-gesting that PBOX-15–induced apoptosis occurs independentof Bcl-2.Previously, we showed that PBOX-6 can induce either caspase-

dependent or caspase-independent apoptosis depending on thecell type (4, 25). To investigate the role of caspases in mediatingPBOX-15–induced apoptosis in CLL cells, we used a series ofspecific pharmacologic inhibitors of caspase activity (Fig. 5B).Pretreatment of cells with the pan-caspase inhibitor z-VAD-fmk significantly inhibited PBOX-15–induced apoptosis (n = 21,P < 0.0001), suggesting a requirement for caspase activity. Inter-estingly, pretreatment with caspase-3 inhibitor, Ac-DMQD-CHO,and caspase-9 inhibitor, Ac-LEHD-CMK, had no significant effect,whereas caspase-8 inhibitor, z-IETD-fmk, significantly abrogatedPBOX-15–induced apoptosis (n = 8, P < 0.003; Fig. 5Bi). Pretreat-ment of the CLL cell line PGA1 with z-VAD-fmk and z-IETD-fmksignificantly inhibited PBOX-15–induced apoptosis (Fig. 5Bii).The tubulin depolymerization agent nocodazole has been shownto induce apoptosis in CLL cells primarily by activation ofcaspase-9 (12), and here, we show that z-VAD-fmk and Ac-LEHD-CMK significantly inhibit nocodazole-induced apoptosisin PGA1 cells. To confirm the requirement for caspase-8 activationin PBOX-15–induced apoptosis, we used siRNA to knock downexpression in PGA1 cells. We achieved partial knockdown ofcaspase-8 and observed significant inhibition of PBOX-15–induced

Figure 4. PBOX-15 is more potent than fludarabine in inducing apoptosis inCLL cells. A, CLL cells isolated from patient #47 were treated with 1 μmol/LPBOX-15 or the indicated concentrations of fludarabine for the indicated timepoints and apoptosis was measured by Annexin V/PI assay. B, CLL cells isolatedfrom 14 patients were treated for 24 h with 1 μmol/L PBOX-15 or the indicatedfludarabine concentrations, and apoptosis was measured as above. PBOX-15induced significantly greater apoptosis than 50 μmol/L fludarabine (P < 0.02).C, CLL cells, harboring a del17p, isolated from three patients were treated eitherwith 1 μmol/L PBOX-15 or with 20 or 50 μmol/L fludarabine and apoptosiswas measured as above.

PBOX-15 Induces Apoptosis in CLL Cells

8371 Cancer Res 2009; 69: (21). November 1, 2009www.aacrjournals.org

Research. on May 8, 2016. © 2009 American Association for Cancercancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 13, 2009; DOI: 10.1158/0008-5472.CAN-09-0131

apoptosis compared with control siRNA–transfected cells(Fig. 5C). In addition, immunoblot analysis of caspase-8 in sixpatient samples undergoing PBOX-15–induced apoptosisshowed down-regulation of full-length procaspase-8 in fiveCLL samples (nos. 15, 20, 26, 28, and 40) and increased detectionof cleaved caspase-8 fragments in three samples (nos. 28, 37, and40), suggesting PBOX-15–induced activation of caspase-8 in thesecells (Fig. 5D).

PBOX-15–induced apoptosis is JNK dependent in low-riskCLL and JNK independent in high-risk CLL. JNK has beenreported to be constitutively activated in CLL (35), and we havepreviously shown a requirement for early activation of JNK in ap-optosis induced by PBOX-6 in several leukemic cell lines (36). Toinvestigate a role for JNK activation in PBOX-15–induced apoptosisin CLL cells, we pretreated cells with the specific JNK inhibitorSP600125 before incubation with PBOX-15 (Fig. 6A). Inhibition

Figure 5. Mechanism of PBOX-15–induced apoptosis in CLL cells. A, CLL cells isolated from four patients were treated with 0.1% ethanol control (−) or 1 μmol/LPBOX-15 (+) for 24 h and Bcl-2 expression was analyzed by immunoblotting. No change in Bcl-2 expression levels or phosphorylation status was detected. K562 cellstreated with 1 μmol/L PBOX-15 for 24 h show a Bcl-2 band shift, which is indicative of Bcl-2 phosphorylation. B, inhibition of PBOX-15 using pharmacologicinhibitors of caspases. i, CLL cells were treated directly with 1 μmol/L PBOX-15 or pretreated for 1 h with specific caspase inhibitors before addition of PBOX-15.Pan-caspase inhibitor, 50 μmol/L z-VAD-fmk; caspase-3 inhibitor, 100 μmol/L z-DMQD-CHO (casp3 inh); caspase-8 inhibitor, 50 μmol/L z-IETD-fmk (casp8 inh);and caspase-9 inhibitor, 50 μmol/L AC-LEHD-CMK (casp9 inh). After 24 h, apoptosis was measured by Annexin V/PI assay. *, P < 0.0001, PBOX-15 versusPBOX-15 + z-VAD-fmk; n = 21. **, P < 0.003, PBOX-15 versus PBOX-15 + casp8 inh; n = 8. ii, PGA1 cells were pretreated with 50 μmol/L of z-VAD-fmk,z-IETD-fmk (casp8 inh), or AC-LEHD-CMK (casp9 inh) for 1 h before treatment with 1 μmol/L PBOX-15 or 1 μmol/L nocodazole (Noz). After 24 h, apoptosiswas measured as above. *, P < 0.005, PBOX-15 versus PBOX-15 + z-VAD-fmk. **, P < 0.005, PBOX-15 versus PBOX-15 + casp8 inh. #, P < 0.0005, Noz versusNoz + z-VAD-fmk. ##, P < 0.002, Noz versus Noz + casp9 inh. n = 3. Columns, mean; bars, SE. C, PGA1 cells were incubated for 72 h with either Accell SMARTpoolcaspase-8 siRNA or Accell nontargeting siRNA pool before addition of 0.5 μmol/L PBOX-15 for 16 h. Caspase-8 expression was assessed by immunoblotting andapoptosis was measured as above. *, P < 0.04; n = 3. Columns, mean; bars, SE. D, CLL cells isolated from six patients were treated with 1 μmol/L PBOX-15 for 24 h.Apoptosis was measured as above and caspase-8 expression was analyzed by immunoblotting. The caspase-8 antibody detects full-length procaspase-8 (57 kDa)and the cleaved caspase-8 fragment (p43/p41), which is indicative of caspase-8 activation.

Cancer Research

8372Cancer Res 2009; 69: (21). November 1, 2009 www.aacrjournals.org

Research. on May 8, 2016. © 2009 American Association for Cancercancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 13, 2009; DOI: 10.1158/0008-5472.CAN-09-0131

of JNK activity significantly inhibited PBOX-15–induced apoptosis(n = 17, P < 0.0005). However, a large heterogeneity in the inhibitionof apoptosis was observed. Interestingly, when samples were sub-grouped according to IgVH mutational status and ZAP-70 expres-sion, it was observed that JNK inhibition completely inhibitedapoptosis in M-IgVH and ZAP-70

− cells (P < 0.0006 and P < 0.001,respectively) while having little effect on U-IgVH and ZAP-70

+ cells(Fig. 6B). As expected, there was a high level of concordance withIgVH mutational status and ZAP-70 expression (eight of nine pa-tients with M-IgVH were ZAP-70− and four of six patients withU-IgVH were ZAP-70+). To confirm this role for JNK activation,we used siRNA to knock down expression in PGA1 (M-IgVH) andHG3 (U-IgVH) cells; however, we were unable to achieve satisfactoryknockdown of JNK using this approach (data not shown). This maybe due to our finding that pharmacologic inhibition of JNK inducedapoptosis in these cell lines (data not shown), suggesting that JNKmay have a role as a survival factor in proliferating CLL cells. Wedetected activation of JNK following treatment with PBOX-15 in sixCLL patient samples by immunoblotting using a phospho-JNKantibody (Fig. 6C). PBOX-15 seemed to selectively activate thep46 JNK isoform because little phosphoylated p54 JNK wasdetected. Increased activation of JNK was detected in CLL cells thatwere either U-IgVH or ZAP-70

+ compared with cells that were M-IgVH and ZAP-70

−.

DiscussionWe have previously shown that PBOX-15, a novel tubulin depo-

lymerization agent, causes disruption of the microtubule network(7) and, similar to another tubulin depolymerization agent, noco-dazole, induces mitotic arrest in G2-M before onset of apoptosis inproliferating cell lines (26). However, microtubules are critical inmany cellular processes as well as cytokinesis, and therefore, disrup-tion of microtubule dynamics is likely to result in significant cellulareffects. We recently showed that PBOX-15 inhibits integrin-associated T-lymphocyte migration (27), and other MTAs have beenshown to have anti-inflammatory properties (13, 37). Microtubulesare involved in B-cell signaling (15), and consequently, microtubuledisruption may influence B-cell survival. This led us to investigatethe activity of PBOX-15 in nonproliferating CLL cells.We showed PBOX-15–induced destruction of microtubule struc-

ture in CLL cells, consistent with our previous findings that PBOX-15 is a tubulin depolymerization agent (7, 27). We observed thisloss of microtubule architecture in all CLL cells treated withPBOX-15, even though in some instances the number of cells un-dergoing apoptosis was less than 50%. This suggests that PBOX-15disrupts microtubules in CLL cells through direct effects ontubulin and not secondary to apoptosis induction. Indeed, ourprevious work has shown decreased tubulin polymerization and

Figure 6. PBOX-15–induced apoptosis in M-IgVH and ZAP-70 negative subgroups of CLL is JNK activation dependent. A, CLL cells were either treated directlywith 1 μmol/L PBOX-15 or pretreated for 1 h with 25 μmol/L SP600125 (JNK inhibitor) before incubation with PBOX-15. Apoptosis was measured after 24 h by the AnnexinV/PI assay. *, P < 0.0005, PBOX-15 versus PBOX-15 + SP600125; n = 17. B, effect of JNK inhibition on PBOX-15–induced apoptosis in CLL cells from patientsgrouped according to (i) mutational status of IgVH genes or (ii) ZAP-70 expression. Columns, mean apoptosis; bars, SE. **, P < 0.0006; ***, P < 0.001; ns, not significant(P > 0.06). C, CLL cells isolated from six patients were treated with 1 μmol/L PBOX-15 for 24 h. Cell lysates were resolved by SDS-PAGE and active JNK wasdetected by immunoblotting using a phospho-JNK (Thr183/Tyr185) antibody.

PBOX-15 Induces Apoptosis in CLL Cells

8373 Cancer Res 2009; 69: (21). November 1, 2009www.aacrjournals.org

Research. on May 8, 2016. © 2009 American Association for Cancercancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 13, 2009; DOI: 10.1158/0008-5472.CAN-09-0131

posttranslational modifications following PBOX-15 treatment inlymphocytes (27).Here, we show that PBOX-15 induced apoptosis in CLL cells,

including those with poor prognostic indicators: U-IgVH, CD38+,

and ZAP-70+. Of particular note is the sensitivity of del17p cellsto PBOX-15, resulting in loss of p53. These cells were resistant tofludarabine-induced apoptosis but exhibited significant apoptosiswhen exposed to PBOX-15. Cells without del17p underwentfludarabine-induced apoptosis, but were more sensitive to PBOX-15–induced apoptosis. These results suggest that fludarabine-induced apoptosis is p53 dependent, whereas PBOX-15–inducedapoptosis is primarily p53 independent. This is a relevant observa-tion in the context of combating fludarabine resistance in high-riskCLL patients.The mechanism of PBOX-15–induced apoptosis in CLL seems to

be different from that of the tubulin depolymerization agent noco-dazole. Here, we show that PBOX-15–induced apoptosis is caspase-8 dependent, whereas nocodazole-induced apoptosis is caspase-9dependent. In addition, nocodazole has been reported to have adifferential effect on Bcl-2 in CLL cells: decreasing Bcl-2 expressionin some samples and inducing phosphorylation in others after aslittle as 1-hour exposure (12). However, we detected no changes inexpression levels or phosphorylation of Bcl-2 following treatmentwith PBOX-15, although we cannot discount the possibility of anearlier transient phosphorylation of Bcl-2 (38).The inhibition of apoptosis by a caspase-8 inhibitor and siRNA

knockdown suggests caspase-8 activation as the primary mecha-nism of PBOX-15–induced apoptosis in CLL. Caspase-8 activationis involved in the death receptor apoptotic pathway; however,earlier evidence has suggested that this pathway is not functionalin CLL. CLL cells have been shown to be resistant to CD95/Fasligand– and tumor necrosis factor–related apoptosis-inducingligand (TRAIL)–induced apoptosis (39, 40), and activation ofcaspase-8 has been shown to be low in B-lymphocytic leukemiacells (41). However, more recently, it has been reported that acti-vation of caspase-8 can induce apoptosis and sensitize CLL cellsto TRAIL-induced apoptosis (42). Activation of a caspase-8–dependent apoptosis pathway in CLL cells by PBOX-15 is potentiallyvery promising as it may overcome chemoresistance to fludarabineand rituximab, which induce apoptosis via activation of caspase-9and the mitochondrial pathway (43, 44).Here, we provide evidence of a role for JNK activation in PBOX-

15–induced apoptosis in ex vivo CLL cells. Our results suggest thatthis PBOX-15 effect is JNK dependent in M-IgVH and ZAP-70

− cellsand JNK independent in U-IgVH and ZAP-70

+ cells. We have previ-ously shown a role for JNK-mediated phosphorylation and in-activation of antiapoptotic Bcl-2 proteins, which is required for

PBOX-6–induced apoptosis in several leukemic cell lines (32). How-ever, the role of JNK in PBOX-15–induced apoptosis in CLL cellsseems to be somewhat different: pharmacologic inhibition of JNKresulted in apoptosis in CLL cell lines, and no evidence of PBOX-15–induced Bcl-2 phosphorylation was seen in ex vivo CLL cells.Although requiring further confirmation, this differential role for

JNK activation in high-risk and low-risk CLL is interesting. Al-though CLL cells have impaired signaling via the B-cell receptorcompared with normal B cells, U-IgVH and ZAP-70

+ CLL cells haveincreased B-cell receptor signaling in response to antigen stimula-tion compared with M-IgVH and ZAP-70

− cells, suggesting that thisplays an important role in the clinical behavior of this disease (16,45). Indeed, downstream JNK, extracellular signal–regulated ki-nase, and Akt activation has been reported to be more prevalentin CLL cells with U-IgVH, compared with M-IgVH, following activa-tion of the B-cell receptor (46), and here, we show greater relativelevels of JNK activation in cells that were either U-IgVH or ZAP-70

+

compared with cells that were M-IgVH and ZAP-70−. Differences in

prosurvival signaling in the subgroups of CLL may be reflected inthe role of JNK in PBOX-15–induced apoptosis shown here. JNKplays a complex role in B-cell survival (47); therefore, this role ofJNK in CLL may have further therapeutic significance.Our data extend the anticancer activity of PBOX-15, a novel

tubulin-targeting agent, to primary CLL cells, indicating its abilityto induce apoptosis in a predominantly nonproliferating popula-tion of tumor cells. Importantly, PBOX-15 induces apoptosis inCLL cells with poor prognostic markers, including fludarabine-resistant cells with del17p, indicating its potential as an anticanceragent in chemoresistant disease. Based on these data, novel tubulin-depolymerizing agents with distinct mechanisms of action, such asPBOX-15, may indicate new therapeutic approaches for treatmentof CLL and other malignancies characterized by low proliferationrates.

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

AcknowledgmentsReceived 1/15/09; revised 8/18/09; accepted 8/28/09; published OnlineFirst 10/13/09.

Grant support: Enterprise Ireland, Cancer Research Ireland, and the HigherEducation Authority of Ireland Programme for Research in Third-Level Institutions.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Professor Anders Rosén (Department of Clinical and ExperimentalMedicine, Linköping University, Linköping, Sweden) for the kind gift of PGA1 andHG3 cell lines, and Dr. Orla Hanrahan (School of Biochemistry and Immunology, TrinityCollege Dublin, Dublin, Ireland) for help with microscopy.

References1. Caligaris-Cappio F, Hamblin TJ. B-cell chronic lym-phocytic leukemia: a bird of a different feather. J ClinOncol 1999;17:399–408.

2. Chiorazzi N. Cell proliferation and death: forgottenfeatures of chronic lymphocytic leukemia B cells. BestPract Res Clin Haematol 2007;20:399–413.

3. Tam CS, Keating MJ. Chemoimmunotherapy of chron-ic lymphocytic leukemia. Best Pract Res Clin Haematol2007;20:479–98.

4. Zisterer DM, Campiani G, Nacci V, Williams DC.Pyrrolo-1,5-benzoxazepines induce apoptosis in HL-60,

Jurkat, and Hut-78 cells: a new class of apoptotic agents.J Pharmacol Exp Ther 2000;293:48–59.

5. Mulligan JM, Campiani G, Ramunno A, Nacci V,Zisterer DM. Inhibition of G1 cyclin-dependent kinaseactivity during growth arrest of human astrocytomacells by the pyrrolo-1,5-benzoxazepine, PBOX-21. Bio-chim Biophys Acta 2003;1639:43–52.

6. Greene LM, Fleeton M, Mulligan J, et al. The pyrrolo-1,5-benzoxazepine, PBOX-6, inhibits the growth ofbreast cancer cells in vitro independent of estrogenreceptor status and inhibits breast tumour growthin vivo. Oncol Rep 2005;14:1357–63.

7. Mulligan JM, Greene LM, Cloonan S, et al. Identifica-tion of tubulin as the molecular target of proapoptoticpyrrolo-1,5-benzoxazepines. Mol Pharmacol 2006;70:60–70.

8. Kavallaris M, Verrills NM, Hill BT. Anticancer therapywith novel tubulin-interacting drugs. Drug Resist Updat2001;4:392–401.

9. Thomson AER, Robinson MA. Cytocidal action of col-chicine in vitro on lymphocytes in chronic lymphocyticleukaemia. Lancet 1967;2:868–70.

10. Wetherley-Mein G, Thomson AE, O'Connor TW,Peel WE, Singh AK. Colchicine ultrasensitivity of

Cancer Research

8374Cancer Res 2009; 69: (21). November 1, 2009 www.aacrjournals.org

Research. on May 8, 2016. © 2009 American Association for Cancercancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 13, 2009; DOI: 10.1158/0008-5472.CAN-09-0131

lymphocytes in chronic lymphocytic leukaemia. Br JHaematol 1983;54:111–20.

11. Vilpo JA, Koski T, Vilpo LM. Selective toxicity ofvincristine against chronic lymphocytic leukemia cellsin vitro. Eur J Hematol 2000;65:370–8.

12. Beswick RW, Ambrose HE, Wagner SD. Nocodazole,a microtubule depolymerising agent, induces apoptosisof chronic lymphocytic leukaemia cells associated withchanges in Bcl-2 phosphorylation and expression. LeukRes 2006;30:427–36.

13. Moon EY, Lerner A. Benzylamide sulindac analoguesinduce changes in cell shape, loss of microtubules andG2-M arrest in a chronic lymphocytic leukemia (CLL)cell line and apoptosis in primary CLL cells. CancerRes 2002;62:5711–9.

14. Zhou Y, Hileman EO, Plunkett W, Keating MJ,Huang P. Free radical stress in chronic lymphocyticleukemia cells and its role in cellular sensitivity toROS-generating anticancer agents. Blood 2003;101:4098–104.

15. Fernandez JA, Keshvara LM, Peters JD, et al. Phos-phorylation- and activation-independent association ofthe tyrosine kinase Syk and the tyrosine kinase sub-strates Cbl and Vav with tubulin in B-cells. J Biol Chem1999;274:1401–6.

16. Lanham S, Hamblin T, Oscier D, et al. Differential sig-naling via surface IgM is associated with V-H genemutational status and CD38 expression in chronic lym-phocytic leukemia. Blood 2003;101:1087–93.

17. Elder ME, Lin D, Clever J, et al. Human severe com-bined immunodeficiency due to a defect in ZAP-70, aT-cell tyrosine kinase. Science 1994;264:1596–9.

18. Chen LG, Apgar J, Huynh L, et al. ZAP-70 directly en-hances IgM signaling in chronic lymphocytic leukemia.Blood 2005;105:2036–41.

19. Campiani G, Nacci V, Fiorini I, et al. Synthesis,biological activity, and SARs of pyrrolobenzoxazepinederivatives, a new class of specific “peripheral-type”benzodiazepine receptor ligands. J Med Chem 1996;39:3435–50.

20. McGee MM, Gemma S, Butini S, et al. Pyrrolo[1,5]benzoxa(thia)zepines as a new class of potent apoptoticagents. Biological studies and identification of an intra-cellular location of their drug target. J Med Chem 2005;48:4367–77.

21. Hayat A, O'Brien D, O'Rourke P, et al. CD38 expres-sion level and pattern of expression remains a reliableand robust marker of progressive disease in chroniclymphocytic leukemia. Leuk Lymphoma 2006;47:2371–9.

22. Crespo M, Bosch F, Villamor N, et al. ZAP-70 expres-sion as a surrogate for immunoglobulin-variable-regionmutations in chronic lymphocytic leukemia. N Engl JMed 2003;348:1764–75.

23. Myhrinder AL, Hellqvist E, Sidorova E, et al. A newperspective: molecular motifs on oxidized LDL, apopto-tic cells, and bacteria are targets for chronic lymphocyt-ic leukemia antibodies. Blood 2008;111:3838–48.

24 . Vermes I , Haanen C , S te f f ens -Nakken H,Reutelingsperger C. A novel assay for apoptosis. Flowcytometric detection of phosphatidylserine expressionon early apoptotic cells using fluorescein labelledAnnexin V. J Immunol Methods 1995;184:39–51.

25. McGee MM, Campiani G, Ramunno A, et al. Pyrrolo-1,5-benzoxazepines induce apoptosis in chronic myeloge-nous leukemia (CML) cells by bypassing the apoptoticsuppressor bcr-abl. J Pharmacol Exp Ther 2001;296:31–40.

26. Greene LM, Campiani G, Lawler M, Williams DC,Zisterer DM. BubR1 is required for a sustained mitoticspindle checkpoint arrest in human cancer cells treatedwith tubulin-targeting pyrrolo-1,5-benzoxazepines. MolPharmacol 2008;73:419–30.

27. Verma NK, Dempsey E, Conroy J, et al. A new micro-tubule-targeting compound PBOX-15 inhibits T-cell mi-gration via post-translational modifications of tubulin.J Mol Med 2008;86:457–69.

28. Pessina A, Malerba F, Gribaldo L. Hematotoxicitytesting by cell clonogenic assay in drug developmentand preclinical trials. Curr Pharm Des 2005;11:1055–65.

29. Gandhi V, Kemena A, Keating MJ, Plunkett W. Cellu-lar pharmacology of fludarabine triphosphate in chroniclymphocytic-leukemia cells during fludarabine therapy.Leuk Lymphoma 1993;10:49–56.

30. Dohner H, Fischer K, Bentz M, et al. P53 gene dele-tion predicts for poor survival and nonresponse to ther-apy with purine analogs in chronic B-cell leukemias.Blood 1995;85:1580–9.

31. Turgut B, Vural O, Pala FS, et al. 17p deletion is as-sociated with resistance of B-cell chronic lymphocyticleukemia cells to in vitro fludarabine-induced apoptosis.Leuk Lymphoma 2007;48:311–20.

32. McGee MM, Greene LM, Ledwidge S, et al. Selectiveinduction of apoptosis by the pyrrolo-1,5-benzoxaze-pine 7-[dimethylcarbamoyl]oxy]-6-(2-naphthyl)pyrrolo-[2,1-d] (1,5)-benzoxazepine (PBOX-6) in leukemia cellsoccurs via the c-Jun NH2-terminal kinase-dependentphosphorylation and inactivation of Bcl-2 and Bcl-XL.J Pharmacol Exp Ther 2004;310:1084–95.

33. Ruvolo PP, Deng X, May WS. Phosphorylation ofBcl2 and regulation of apoptosis. Leukemia 2001;15:515–22.

34. Thomas A, Pepper C, Hoy T, Bentley P. Bryostatininduces protein kinase C modulation, Mcl-1 up-regulation and phosphorylation of Bcl-2 resulting incellular differentiation and resistance to drug-inducedapoptosis in B-cell chronic lymphocytic leukemia cells.Leuk Lymphoma 2004;45:997–1008.

35. Poggi A, Catellani S, Bruzzone A, et al. Lack of theleukocyte-associated Ig-like receptor-1 expression inhigh-risk chronic lymphocytic leukaemia results in theabsence of a negative signal regulating kinase activationand cell division. Leukemia 2008;22:980–8.

36. McGee MM, Campiani G, Ramunno A, et al. Activa-tion of the c-Jun N-terminal kinase (JNK) signaling path-way is essential during PBOX-6-induced apoptosis inchronic myelogenous leukemia (CML) cells. J Biol Chem2002;277:18383–9.

37. Mirzapoiazova T, Kolosova IA, Moreno L, et al. Sup-pression of endotoxin-induced inflammation by Taxol.Eur Respir J 2007;30:429–35.

38. Du LH, Lyle CS, Chambers TC. Characterization ofvinblastine-induced Bcl-xL and Bcl-2 phosphorylation:evidence for a novel protein kinase and a coordinatedphosphorylation/dephosphorylation cycle associatedwith apoptosis induction. Oncogene 2005;24:107–17.

39. Romano C, De Fanis U, Sellitto A, et al. Induction ofCD95 upregulation does not render chronic lymphocyt-ic leukemia B-cells susceptible to CD95-mediated apo-ptosis. Immunol Lett 2005;97:131–9.

40. Secchiero P, Tiribelli M, Barbarotto E, et al. Aberrantexpression of TRAIL in B chronic lymphocytic leukemia(B-CLL) cells. J Cell Physiol 2005;205:246–52.

41. Kang J, Kisenge RR, Toyoda H, et al. Chemical sensi-tization and regulation of TRAIL-induced apoptosis in apanel of B-lymphocytic leukaemia cell lines. Br J Hema-tol 2003;123:921–32.

42. Lagneaux L, Gillet N, Stamatopoulos B, et al. Valproicacid induces apoptosis in chronic lymphocytic leukemiacells through activation of the death receptor pathwayand potentiates TRAIL response. Exp Hematol 2007;35:1527–37.

43. Genini D, Adachi S, Chao Q, et al. Deoxyadenosineanalogs induce programmed cell death in chronic lym-phocytic leukemia cells by damaging the DNA and bydirectly affecting the mitochondria. Blood 2000;96:3537–43.

44. Byrd JC, Kitada S, Flinn IW, et al. The mechanism oftumor cell clearance by rituximab in vivo in patientswith B-cell chronic lymphocytic leukemia: evidence ofcaspase activation and apoptosis induction. Blood2002;99:1038–43.

45. Kipps TJ. The B-cell receptor and ZAP-70 in chroniclymphocytic leukemia. Best Pract Res Clin Hematol2007;20:415–24.

46. Petlickovski A, Laurenti L, Li XP, et al. Sustained sig-naling through the B-cell receptor induces Mcl-1 andpromotes survival of chronic lymphocytic leukemia Bcells. Blood 2005;105:4820–7.

47. Niiro H, Clark EA. Regulation of B-cell fate byantigen-receptor signals. Nat Rev Immunol 2002;2:945–56.

PBOX-15 Induces Apoptosis in CLL Cells

8375 Cancer Res 2009; 69: (21). November 1, 2009www.aacrjournals.org

Research. on May 8, 2016. © 2009 American Association for Cancercancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 13, 2009; DOI: 10.1158/0008-5472.CAN-09-0131

2009;69:8366-8375. Published OnlineFirst October 13, 2009.Cancer Res   Anthony M. McElligott, Elaina N. Maginn, Lisa M. Greene, et al.   Prognostic Subgroups of Chronic Lymphocytic LeukemiaPyrrolo-1,5-Benzoxazepine-15 Induces Apoptosis in Poor The Novel Tubulin-Targeting Agent

  Updated version

  10.1158/0008-5472.CAN-09-0131doi:

Access the most recent version of this article at:

  Material

Supplementary

  ml

http://cancerres.aacrjournals.org/content/suppl/2009/10/13/0008-5472.CAN-09-0131.DC1.htAccess the most recent supplemental material at:

   

   

  Cited articles

  http://cancerres.aacrjournals.org/content/69/21/8366.full.html#ref-list-1

This article cites 47 articles, 19 of which you can access for free at:

  Citing articles

  http://cancerres.aacrjournals.org/content/69/21/8366.full.html#related-urls

This article has been cited by 1 HighWire-hosted articles. Access the articles at:

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  .permissions@aacr.orgDepartment at

To request permission to re-use all or part of this article, contact the AACR Publications

Research. on May 8, 2016. © 2009 American Association for Cancercancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 13, 2009; DOI: 10.1158/0008-5472.CAN-09-0131