Aplidine, a New Anticancer Agent of Marine Origin, Inhibits Vascular Endothelial Growth Factor (VEGF) Secretion and Blocks VEGF

Leukemia (2003) 17, 52–59  2003 Nature Publishing Group All rights reserved 0887-6924/03 $25.00 www.nature.com/leu Aplidine, a new anticancer agent of marine origin, inhibits vascular endothelial growth factor (VEGF) secretion and blocks VEGF-VEGFR-1( ﬂt-1) autocrine loop in human leukemia cells MOLT-4 M Broggini1, SV Marchini1, E Galliera1, P Borsotti2, G Taraboletti2, E Erba2, M Sironi2, J Jimeno3, GT Faircloth4, R Giavazzi2 and M D’Incalci1

1Laboratory of Molecular Pharmacology, Istituto di Ricerche Farmacologiche ‘Mario Negri’, Milan, Italy; 2Laboratory of Biology and Treatment of Metastasis, Department of Oncology, Istituto di Ricerche Farmacologiche ‘Mario Negri’, Milano and Bergamo, Italy; 3PharmaMar SA, Clinical R&D, Tres Cantos, Madrid, Spain; and 4PharmaMar, Research & Development, Cambridge, MA, USA

The mechanism by which aplidine, a marine natural product in early clinical development as an anticancer agent, induces cell growth inhibition and apoptosis has been investigated in the human leukemia cell line MOLT-4. This cell line is characterized not only by the ability to secrete VEGF, but also for the presence on its surface of the VEGF receptor-1 (VEGFR-1). Previous studies from our laboratory concerned with evaluating early changes in gene expression induced by aplidine in MOLT-4 cells have shown that the drug decreases the expression of VEGFR-1 (Marchini et al. Proc Am Assoc Cancer Res 2000; 41: 833). Here, we report the ability of aplidine to block the VEGF/VEGFR-1 loop. We found that aplidine blocked VEGF secretion that was temporally followed by a decrease in both VEGF and VEGFR-1 production. Aplidine did not directly affect either VEGF transcription or stabilization of its mRNA. Trans- fection of MOLT-4 cells with an antisense VEGF cDNA construct, resulted in inhibition of colony formations. One clone, transfected with sense VEGF cDNA, secreting 8–10 times more VEGF than parental cells, was less sensitive to aplidine- induced cytotoxicity and apoptosis than control cells. More- Figure 1 Chemical structure of aplidine. over, addition of VEGF in the medium decreased the activity of aplidine in MOLT-4 cells. These data demonstrate that aplidine inhibits the growth and induces apoptosis in MOLT-4 cells This receptor is a member of a family of receptors able to through the inhibition of VEGF secretion which blocks the VEGF/VEGFR-1 autocrine loop necessary for the growth of bind the angiogenic factor VEGF (vascular endothelial growth these cells. factor), and is normally present on the surface of vascular Leukemia (2003) 17, 52–59. doi:10.1038/sj.leu.2402788 endothelial cells.13–17 Increasing evidence suggests, however, Keywords: VEGF; marine compounds; autocrine loop; leukemia; that certain cancer cells of different origin, including leukemic apoptosis cells, also express VEGF receptors on their surface.18–23 On the other hand, VEGF is secreted by virtually all cancer cells as a homodimeric glycoprotein which is able to bind VEGFR- Introduction 1 and other components of the VEGF receptors family with high affinity.14,24 The simultaneous presence of a receptor and Aplidine, dehydrodidemnin B (see structure in Figure 1) is a the ability to secrete the ligand for it, suggests the possible new marine depsipeptide isolated from Aplidium albicans, presence of an autocrine loop important for the growth of can- shows strong antitumor activity against different human cancer cells expressing these receptors. In different experimental cer cells growing in vitro and in vivo.1–5 It has completed systems it has been shown that VEGF can act as an autocrine phase I trials and is under phase II development.6–9 Although factor.25–29 Furthermore, VEGF has been shown to act as a its precise mechanism of action is still unknown, aplidine is survival factor inhibiting apoptosis through the expression of able to induce rapid p53-independent apoptosis in different antiapoptotic proteins.17,30,31 cancer cell lines growing in vitro.10,11 It has also been shown In this study we present evidence of a strong effect of aplid- that the drug induces a cell cycle perturbation with a block ine on the VEGF/VEGFR-1 autocrine loop regulating the of human leukemic MOLT-4 cells mainly in G1 phase of the growth of the human leukemic cell line MOLT-4. These find- cell cycle.10,11 Studies on changes in gene expression induced ings indicate one of the mechanisms by which the drug is by aplidine in this cell line revealed a strong alteration of the able to induce growth arrest and apoptosis in cancer cells thus pattern of gene expression at early times after treatment.12 providing a clinically relevant mechanistic insight. Among these changes is a clear and reproducible decrease in the expression of the VEGFR-1 (vascular endothelial growth factor-receptor 1, flt-1). Materials and methods

Cells, clones and treatment Correspondence: M Broggini, Laboratory of Molecular Pharmacology, Istituto di Ricerche Farmacologiche ‘Mario Negri’, via Eritrea 62, The human leukemic cell line MOLT-4, derived from a patient 20157 Milan, Italy; Fax: 39-023546277 suffering from T-lymphoblastic leukemia relapsed after multid- Received 17 June 2002; accepted 17 September 2002 rug chemotherapy,32 was maintained in RPMI 1640 sup- The marine compound aplidine blocks VEGF secretion M Broggini et al

␮ 53 plemented with 10% FCS. Exponentially growing cells were MgCl2, 0.1% NP40, 5% sucrose, 1 g poly(dIdC), and 1 ng treated for 1 h with 0, 1, 5, 10 or 20 nM of aplidine. Cell of32P-end-labeled oligonucleotide, in the absence or presence counts were taken after 24, 48 and 72 h of incubation in drug- of different aplidine concentrations. free medium after staining with erythrosin. Activity of aplidine The oligonucleotides utilized, HIF-1 (5Ј-TGCATACGTGGG in the presence of exogenous VEGF was tested by adding 10 CTCCAACAG-3Ј) and AP-1 (5Ј-AGGGGCAAAGTGAGTGAC ng/ml of recombinant human VEGF (R&D Systems, Minnea- CTGCTT-3Ј), have been synthetized from their recognition polis, MN, USA) every 24 h in the medium. These experiments sequence present in the VEGF promoter. DNA–protein com- were performed in low-serum concentration (1%), to minim- plexes were separated by electrophoresis through a 5% native ize the effects of grow factors present in the serum. polyacrylamide gel, dried and visualized by phosphoimager Human umbilical vein endothelial cells (HUVEC, kindly analysis (Storm, Amersham Biosciences, Milan, Italy). provided by Dr Breviario, Mario Negri Institute, Milan, Italy) were used as cells not secreting VEGF. Adherent cells were treated for 1 h with 5 or 20 nM aplidine and total mRNA was extracted 6 and 24 h after treatment using the SV Total RNA kit (Promega, Milan, Italy). MOLT-4 cells were transfected with the human VEGF cDNA, both in sense and antisense orientation (kindly sup- plied by M Presta, University of Brescia, Italy) by electroporation. Seventy-two hours after transfection, cells were seeded in 96-well plates at the density of 0.5 cells/well in medium containing 500 ␮g/ml of G418 used as selection antibiotic. The number of colonies formed were quantiﬁed after approximately 14 days. As a control, human ovarian cancer cells A2780 were transfected by calcium phosphate precipitation using the same constructs. This cell line was selected as a negative control because of the lack of expression of VEGF receptors and because it had already been successfully used in our laboratory in transient and stable transfection experiments.33,34 Colonies were stained with crystal violet after 14 days in selection medium. One MOLT-4-derived clone, overexpressing VEGF, was used in subsequent studies and was cultured in RPMI 1640 in the presence of 500 ␮g/ml of G418.

VEGF protein levels

Intracellular VEGF protein levels, and the amount secreted in the medium by MOLT-4 cells, was measured with a Quanti- kine immunoassay kit (R&D Systems) according to the manu- facturer’s instructions. aplidine did not interfere with the measurement of VEGF (absorbance of 300 pg/ml VEGF was 0.734 ± 0.008 without aplidine, and 0.710 ± 0.001 in the presence of 20 nM aplidine).

VEGF and VEGFR-1 mRNA analysis

VEGF and VEGFR-1 mRNA were measured by RNase protection using a commercially available kit (Becton Dickinson, Franklin Lakes, NJ, USA) or by Northern blotting analysis according to standard procedures.35

Electrophoretic mobility shift assay

Untreated or aplidine-treated cells (106) were lysed in chilled buffer containing 10 mM Hepes pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 0.8% Non- idet NP-40. Nuclei were pelleted, extracted for 1 h in extrac- tion buffer (20 mM Hepes pH 7.9, 0.4 M NaCl, 1 mM EDTA, Figure 2 Panel (a) VEGF (empty columns) and VEGFR-1 (ﬁlled 1 mM EGTA, 1 mM DTT, 1 mM PMSF) and cleared from columns) mRNA levels in MOLT-4 cells at different times after 1 h ␮ treatment with 20 nM aplidine. Data have been obtained by densito- debris by centrifugation at 12 000 g for 15 min. Ten gof metric analysis. Panel (b) Levels of VEGF secreted in the medium of nucleic extracts were incubated on ice for 1 h in 15 ␮lof untreated or aplidine-treated (20 nM) cells. VEGF was measured by buffer containing 20 mM Hepes pH 7.5, 100 mM NaCl, 1 mM ELISA at different time-points after 1 h drug treatment.

Leukemia The marine compound aplidine blocks VEGF secretion M Broggini et al 54 Measurement of VEGF half-life Milan, Italy) for 60–90 min at 37°C in the dark. After washing in PBS the cells were resuspended in PBS and analyzed by VEGF mRNA half-life was determined by RNase protection. FacsCalibur instrument. At least 10 000 cells were evaluated Total RNA from untreated or aplidine-treated (20 nM) MOLT- for each sample. 4 cells was extracted at 10, 20, 40, 60 and 120 min after the addition of 2.5 ␮g/ml of actinomycin D (Sigma, St Louis, MO, USA). For each time-point, three different replicates were Results performed. The human leukemic cell line MOLT-4 is sensitive to treat-

ment with nanomolar concentrations of aplidine. The IC50 VEGF promoter activity (concentration inhibiting the growth by 50%) of the drug, evaluated after 72 h incubation in drug-free medium following A 1005-bp long DNA fragment containing the promoter 1 h treatment, is approximately 10 nM. region of VEGF, subcloned 5Ј to the luciferase gene in the In a previous study aimed at evaluating early changes in PGL2 basic vector (Promega), was used to analyze transcrip- gene expression in MOLT-4 cells after 1 h treatment with 10 tion control of this gene under varying treatment conditions. nM of aplidine, we found that among different genes whose Cells were co-transfected by electroporation with 4 ␮gof expression was modulated, a time-dependent reduction of the pGL2-derived plasmids containing the 5Ј flanking region of VEGF-R1 mRNA was observed.12 This decrease was con- the VEGF gene and 0.05 ␮g of pRL-SV40 used for internal firmed by PCR and subsequently by Western blotting analy- normalization. After treatment with varying aplidine concen- sis.12 To study whether the changes in gene expression trations for 1 h, reporter gene activities were evaluated 24 h induced by aplidine could be extended to other members of after using the Dual Luciferase system (Promega). Results are the VEGF receptor and to ligands of VEGFR-1, we used the expressed as the percentage of the control luciferase activity RNase protection assay with probes of different genes normalized by the renilla activity value used as internal stan- involved in angiogenesis including VEGF. The results (Figure dard. The mean ± s.d. of three independent experiments is 2a) clearly show that aplidine, as previously found, induces a shown. decrease in the expression of VEGFR-1, but more dramatically reduces the expression of VEGF mRNA which was not detect- able at 6 and 24 h. VEGFR-2 is not expressed by MOLT-4 Evaluation of apoptosis cells. We therefore tested the effect of aplidine treatment on the At different time intervals after drug washout, the cells were amount of VEGF protein secreted in the medium (Figure 2b). fixed in ethanol 70% and stored at 4°C. The fixed cells were Untreated cells secrete approximately 1.6 pg of VEGF/ml/105 washed in cold PBS and permeabilized with 0.25% Triton X- cells. After treatment with 20 nM aplidine, the levels of VEGF 100 (Sigma) in PBS for 5 min on ice. After removing Triton X- were below the detection limit already at the end of treatment 100, the cells were incubated in 50 ␮l of a solution containing and remained at these levels after 6 and 24 h of post-drug Terminal-dUTP-Transferase and FITC-conjugated dUTP treatment incubation. deoxynucleotides 1:1 in storage buffer (Roche Diagnostic, Figure 3 shows the levels of VEGF secreted in the medium

Figure 3 Dose-dependent decrease in VEGF secretion induced by aplidine treatment of MOLT-4 cells. Cells were treated for 1 h with aplidine, washed and incubated in drug-free medium for a further 6 and 24 h.

Leukemia The marine compound aplidine blocks VEGF secretion M Broggini et al 55 in MOLT-4 cells treated for 1 h with different aplidine concentrations. A clear dose-dependent reduction of VEGF secretion was observed at the end of treatment. At concentrations close to the IC50 the secretion of VEGF remained low 6 and 24 h after drug washout, while at lower concentrations the decrease in VEGF secretion was reversible. We focused on the mechanisms responsible for the aplidine-induced reduction of VEGF production in MOLT-4 cells, initially by checking whether a reduced transcription of the gene could account for this effect. In transient transfection experiments, we used a fragment of the human VEGF promoter containing the first 1000 bp 5Ј to the transcription start- ing site fused to the reporter luciferase gene. Cells were transfected by electroporation, treated for 1 h with different concentrations of aplidine and luciferase activity was determined 24 h after. At all the concentrations tested (all previously reported to induce a down-regulation of VEGF mRNA and protein) the promoter activity was substantially unaffected by aplidine (Figure 4a). The fragment of the promoter we used contains the binding site for HIF-1, one of the most important regulator of VEGF transcription under hypoxic conditions, and for AP-1. By gel retardation assay we analyzed whether aplidine interfered with the binding of HIF-1 and AP-1 to their DNA consensus sequences. When nuclear extracts obtained from MOLT-4 cells were preincubated with different concentrations of aplidine (ranging from 1 nM to 100 ␮M), no change in the binding of either HIF-1 or AP-1, even at the highest concentration was observed (Figure 4b and c). Likewise, aplidine was unable to interfere with the binding of these two transcription factors to their consensus DNA sequences when extracts from cells treated with different concentrations of aplidine were used in gel retardation experiments (data not shown). The levels of VEGF mRNA are not only controlled transcriptionally, but also post transcriptionally. We therefore analyzed whether aplidine was able to change the half-life of VEGF mRNA in MOLT-4 cells (Figure 5). Analysis of VEGF mRNA levels in untreated or aplidine-treated MOLT-4 cells at 0, 20, 40, 60 and 120 min after the addition of actinomycin D Figure 4 Panel (a) Effect of different aplidine concentrations on the showed that the drug, in the three independent experiments activity of a 1000-bp VEGF promoter fragment transiently transfected performed, does not induce a faster degradation of VEGF in MOLT-4 cells. Panel (b) Effect of different aplidine concentrations mRNA. on the binding of HIF-1 to its consensus DNA sequence. 32P-labeled When we measured the intracellular VEGF protein levels, DNA was incubated with MOLT-4 nuclear extracts in the absence (C) or presence of aplidine for 1 h. S and U represent incubation of however, we found that concomitant with a reduction of nuclear extracts and DNA in the presence of a 50-fold molar excess VEGF secreted in the medium, a concentration-dependent of cold specific (S) or unspecific (U) competitor. Panel (c) is the same accumulation of intracellular VEGF was observed at the end as (b) except for the 32P-labeled DNA which contained the AP-1 con- of 1 h of aplidine treatment (Figure 6). sensus DNA sequence instead of the HIF-1 consensus sequence. To test whether the aplidine-induced VEGFR-1 down-regulation was also observable in the absence of VEGF production, we performed additional experiments using normal endo- (with a percentage similar to that obtained after transfection thelial cells, which do express VEGFR-1 but do not secrete with the empty vector), while the transfection of the antisense VEGF. Figure 7 shows that treatment of these cells with aplid- VEGF construct resulted in complete inhibition of colony for- ine did not result in significant inhibition of VEGF-R1 mRNA, mation. Transfection of the same DNA constructs in another even at 20 nM. human cancer cell line (A2780) not expressing VEGF recep- To analyze whether aplidine could exert its antiproliferative tors, did show formation of colonies with both constructs at activity on MOLT-4 by blocking the autocrine VEGF–VEGFR- comparable levels (data not shown). 1 loop, we performed cell growth inhibition experiments in One of the clones transfected with VEGF cDNA was used the presence of exogenous VEGF added in the medium. Ten for subsequent experiments. The increased secretion of VEGF ng/ml of VEGF added in the medium together with aplidine, from this clone resulted in the protection from aplidine treat- and maintained in a drug-free medium incubation period, ment. The drug, in fact, required more than two-fold higher reduced the activity of the drug, particularly at low aplidine concentrations to induce the same growth inhibition as in the concentrations (Figure 8). parental MOLT-4 cells (Figure 9a), and showed a decreased To study further the importance of VEGF secretion for the apoptotic effect, evaluated by flow cytometry after Tunel growth of MOLT-4 cells, we transfected these cells with VEGF staining (Figure 9b). The latter effect is particularly evident cDNA either in sense or antisense orientation. Transfection of after 10 nM, where the percentage of apoptotic cells in par- VEGF sense cDNA allowed us to recover different colonies ental MOLT-4 cells was approximately 90% which decreased

Leukemia The marine compound aplidine blocks VEGF secretion M Broggini et al 56

Figure 5 VEGF mRNA half-life determined in untreated (I) or aplidine-treated (20 nM for 1 h) (L) MOLT-4 cells. Total VEGF RNA was determined at different time-points after the addition of actinomycin D by RNase protection and densitometric analysis of the results.

Figure 7 Northern blot analysis of VEGF-R1 expression in HUVEC cells at different times after treatment with 5 or 20 nM of aplidine. The lower panel reports the ethidium bromide staining of ribosomal RNAs as internal control.

Figure 6 Intracellular VEGF protein levels in MOLT-4 cells treated with different aplidine concentrations for 1 h. Lysates were prepared at the end of treatment and VEGF content measured by ELISA.

to approximately 50% in the clone transfected with VEGF. Both parental and VEGF-transfected MOLT-4 cells grew in vitro at a similar rate, and the actual number of cells of untreated controls 48 h after treatment were 710 000 ± 53 000 and 761 000 ± 89 000 cells/ml respectively for parental and VEGF-transfected cells. Figure 8 Effect of exogenous VEGF on aplidine-induced cytotoxicity in MOLT-4 cells. Cells were treated for 1 h with 1 or 10 nM aplid- Discussion ine in the presence or absence of 10 ng/ml of recombinant VEGF. Cytotoxicity was assessed 96 h after the end of aplidine treatment. Values represent the mean ± s.d. of three independent experiments In this present paper we show that VEGF acts as a growth each consisting of three replicates. factor in MOLT-4 cells and that aplidine, a new and promising anticancer agent currently in clinical trials, 6–9 exerts its cytotoxic activity in these cells by interfering with the autocrine antisense VEGF cDNA resulted in a complete block of the loop involving VEGF and its receptor, VEGFR-1. growth of MOLT-4 cells, is clear and direct evidence that MOLT-4 cells, in fact, not only secrete VEGF as almost all VEGF is acting as a growth factor for this cell line in vitro. cancer cells do, but also express on their surface the VEGFR-1 These results are in agreement with previous ﬁndings showing receptor.11,36 The ﬁnding reported here that transfection with that the use of a soluble VEGFR-1 was able to block the

Leukemia The marine compound aplidine blocks VEGF secretion M Broggini et al 57

Figure 9 Panel (A) Aplidine induced cytotoxicity in parental MOLT-4 cells (black columns) and in a clone which overexpresses human VEGF cDNA (grey columns). Aplidine treatment was for 1 h and cytotoxicity was assessed 72 h after the end of treatment. Values represent the mean ± s.d. of three independent experiments each consisting of three replicates. Panel (b) Representative ﬂow cytometric analysis of TUNEL-positive, apoptotic cells after treatment for 1 h with different doses of aplidine as indicated in MOLT-4 parental cells (B) and VEGF-transfected MOLT- 4 cells (A). Analysis was performed 48 h after drug wash out. growth of MOLT-4 cells.36,37 A role for VEGF as a growth fac- is at present unknown. It seems, however, that this is the pri- tor has been described for endothelial cells and only recently mary effect of the drug which then presumably causes a cas- for certain cancer cells growing in vitro.25–29 In normal endo- cade of events, including inhibition of new VEGF mRNA syn- thelial cells, VEGF stimulates cell growth by inhibiting thesis and down-regulation of VEGFR-1 receptor. That the apoptosis, mainly through the activation of antiapoptotic pro- decrease in the receptor levels is not a direct drug effect but teins.17,30,31 Even if not formally demonstrated, there is evi- rather a consequence of a block in VEGF secretion is also dence, presented here and reported in the literature,26,31 sug- supported by the evidence that human endothelial cells grow- gesting that in certain cancer cells VEGF could also act as ing in culture, not producing VEGF, do not show any decrease a growth factor by inducing antiapoptotic proteins and more in VEGFR-1 levels after treatment with aplidine. Preliminary generally by inhibiting apoptosis. The evidence that a MOLT- evidence suggests that aplidine blocks VEGF secretion with a 4-derived clone overproducing VEGF is less susceptible to certain degree of speciﬁcity being, in another leukemic cell aplidine treatment and is less prone to undergo apoptosis, is line (NB4), able to block VEGF secretion (as observed in in line with this hypothesis. Indirectly, these results suggest MOLT-4 cells) but not the secretion of other proteins such as that compounds able to block VEGF action in these systems TNF-alpha or MCP-1 (data not shown). should arrest all growth and induce apoptosis. The mechanism proposed here (Figure 10), could represent Aplidine, at least in MOLT-4 cells, appears to act by block- one of the possible mechanisms by which aplidine exerts its ing VEGF secretion, a mechanism of action that has never anticancer activity, but it does not exclude the possibility that been described before for an antileukemic or anticancer in other cancer cell types the drug could act with a different agent. mechanism(s). The mechanism by which aplidine inhibits VEGF secretion Finally, VEGF is an important mediator of angiogenesis,

Leukemia The marine compound aplidine blocks VEGF secretion M Broggini et al 58

Figure 10 Proposed scheme for the VEGF/VEGFR-1 autocrine loop and the way in which aplidine induces growth arrest and apoptosis.

being mitogenic for endothelial cells and causing angiogenic other week (q2w) in patients (pts) with solid tumor (ST) and lym- response in vivo.24,38 Recently, an important role for angiog- phoma (NHL). Proc 37th ASCO Annual Meeting, San Francisco, enesis in patients with leukemia has been shown,39–43 raising May 12–15 2001; 20: 120a. 7 Paz-Ares L, Anthony A, Pronk L, Twelves C, Alonso S, Cortes- the possibility of a role of anti-angiogenic drugs in the treat- Funes H, Celli N, Gomez C, Lopez-Lazaro L, Guzman C, Jimeno ment of these types of cancer. By blocking VEGF secretion, J, Kaye S. Phase I clinical and pharmacokinetic study of aplidine, aplidine, apart from its ability to induce growth factor depri- a new marine didemnin, administered as a 24-hour infusion vation, could affect angiogenesis in hematopoietic malig- weekly. Proc 11th NCI-EORTC-AACR Symposium, Amsterdam, nancies. The levels of aplidine that reduced VEGF in our November 7–10 2000; 11: 86. experiments in vitro are in the same range of the levels 8 Maroun J, Belanger K, Seymour L, Soulieres D, Charpentier D, 44 Goel R, Stewart D, Tomiak E, Jimeno J, Matthews S. Phase I study reached in plasma of treated patients. Considering that apli- of aplidine (APL) in a 1 hour daily infusion × 5 weeks in patients dine has shown some activity in patients with lymphomas in (pts) with solid tumors and low and intermediate grade non Hodg- the clinic44 our ﬁnding may have important implications in kin’s lymphomas: a National Cancer Institute of Canada–Clinical explaining this activity. The potential for anti-angiogenic Trials Group (NCIC-CTG) study. Proc of the European Society for activity of aplidine, warrants further investigations. Medical Oncology Meeting, Hamburg, October 13–17 2000; 134. 9 Bowman A, Izquierdo M, Jodrell D, Martinez M, Cicchella B, Jimeno J, Guzman C, Germa-Lluch J, Celli N, Smyth J. Phase I clinical and pharmacokinetic (PK) study of the marine compound Acknowledgements aplidine (APL), administered as a 1 hour weekly infusion. Proc 37th ASCO Annual Meeting. San Francisco, May 12–15 2001; This work was partially supported by a grant ICS 030.1/RF 20; 120a. 00.192 from Ministero della Sanita`, by FIRC and by CNR- 10 Erba E, Bassano L, Di Liberti G, Muradore I, Chiorino G, Ubezio MIUR. P, Vignati S, Codegoni A, Desiderio AM, Faircloth G, Jimeno J, D’Incalci M. Cell cycle phase perturbations and apoptosis induced by aplidine. Br J Cancer 2002; 86: 1510–1511. 11 Erba E, Ronzoni S, Bergamaschi D, Bassano L, Desiderio AM, Fair- References cloth G, Jimeno J, D’Incalci M. Mechanism of antileukemic activity of Aplidine. Proc Am Assoc Cancer Res 1999; 40:3. 1 Faircloth JG, Rinehart K, Nunezde Castro I, Jimeno J. 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