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Original Paper

Acta Haematol 2008;120:51–56 DOI: 10.1159/000158577

In vitro Effects of the Farnesyltransferase Inhibitor Tipifarnib on Myelodysplastic Syndrome Progenitors

Ioannis Kotsianidis a Ioanna Bazdiara a Athanasios Anastasiades a

Emmanouil Spanoudakis a Despoina Pantelidou a Dimitrios Margaritis a

George Bourikas a Roland De Coster b Peter De Porre b Costas Tsatalas a

a Department of Haematology, Democritus University of Thrace, Medical School, Alexandroupolis , Greece; b Johnson and Johnson Pharmaceutical Research and Development, Beerse , Belgium

The leukemic clone is more susceptible in tipifarnib than normal progenitors. Since myelosuppression represents the main obstacle in the clinical use of tipifarnib in MDS, further reduction of the currently employed dose will potentially re-sult in a more tolerable regimen without compromising its antileukemic action. Copyright © 2008 S. Karger AG, Basel

Introduction

The term myelodysplastic syndrome (MDS) encom-passes a heterogeneous group of clonal hematopoietic disorders characterized by ineffective hematopoiesis and variable degrees of transformation into acute leukemia [1] . Despite the fact that MDS represents the most com-mon myeloid malignancy in the elderly, the current ther-apeutic modalities are far from satisfactory. With the ex-ception of lenalidomide [2] in 5q– syndrome, only aza-cytidine [3] and decitabine [4] have shown activity in MDS, leading to recent approval by the USA Food and

Key Words

Farnesyltransferase inhibitors � Hematopoiesis � Molecular-targeted therapy � Myelodysplastic syndrome � Tipifarnib

Abstract

Background: Farnesyltransferase inhibitors (FTIs) target proteins needing prenylation for functioning. Tipifarnib (Zarnestra � ), a potent and specific inhibitor of farnesyltrans-ferase, showed considerable activity in phase I and II studies in myelodysplastic syndrome (MDS), but the optimal regi-men achieving high response rates with minor myelosup-pression remains to be determined. Additionally, a direct ef-fect on purified human MDS progenitors has not yet been shown. Methods: MDS and normal CD34+ cells isolated by using immunomagnetic beads were plated for short-term cultures in semisolid media or liquid cultures for flow-cyto-metric assessment of apoptosis in the presence of either DMSO or various FTI concentrations. Results: Tipifarnib ex-erted selective in vitro toxicity against clonal MDS hemato-poiesis at concentrations less than 10 n M the effect being more prominent in white cell progenitors. This action was not due to apoptosis induction as both normal and MDS progenitors displayed equivalent DiOC3 and annexin V ex-pression up to 72 h after exposure to tipifarnib. Conclusion:

Received: February 18, 2008 Accepted after revision: July 3, 2008 Published online: September 30, 2008

Ioannis Kotsianidis, MD, PhD Department of Haematology, Democritus University of Thrace, Medical School University Hospital of Alexandroupolis GR–68100 Alexandroupolis (Greece) Tel. +30 255 103 0320, Fax +30 255 107 6154, E-Mail ikotsian@med.duth.gr

© 2008 S. Karger AG, Basel0001–5792/08/1201–0051$24.50/0

Accessible online at:www.karger.com/aha

R. De Coster and P. De Porre are employed by the company whose product was studied in the present work.

Kotsianidis et al. Acta Haematol 2008;120:51–5652

Drug Administration. Nevertheless, their efficacy is modest and treatment remains largely palliative in the majority of the patients, whereas a small minority are el-igible for stem cell transplantation which is currently the only curative therapy. Mutations of the Ras family of pro-tooncogenes have been implicated in the molecular pathogenesis of the myeloid malignancies [5] . Attach-ment of ras to the cell membrane, a critical posttransla-tional step that is catalyzed by farnesyltransferase (FTase), is essential in order to become fully functionally active. Beyond Ras activation, FTase participates in multiple cel-lular functions such as cell signaling, proliferation and differentiation, thus regulating normal and cancer cell growth [6] . The above processes are targeted by farnesyl-transferase inhibitors (FTIs), a recently developed group of pharmaceutical compounds that are currently evalu-ated as anticancer agents [5–7] . Compelling data support a role for Ras, but also RhoB, nuclear lamin proteins and centromere-associated proteins in the antitumor effects of FTIs [8, 9] . Tipifarnib (Zarnestra � ) belongs to the group of nonpeptidomimetics FTIs and is able to exert potent inhibition of farnesyltransferase in a variety of tu-mor cell lines and primary cells [10] .

Based on the fact that activating Ras mutations occur in up to 20% of MDS patients [11] several investigators have studied the activity of tipifarnib in MDS patients. However, although the first results are encouraging, the antineoplastic mechanism of tipifarnib is still not well understood and both antiproliferative and preapoptotic mechanisms are implicated [10] . Moreover, to our knowl-edge, a comparison of the in vitro activity of tipifarnib on purified human MDS and normal progenitors has not yet been addressed.

In the present study we sought to investigate the direct effect of tipifarnib on the plating efficiency of selected CD34+ progenitors from patients with MDS and nonma-lignant disorders (NMD). Additionally, in a liquid cul-ture system of the same samples we assessed the tipifarn-ib-induced early apoptosis by measuring the reduction of mitochondrial transmembrane potential and annexin V expression on mature and immature myeloid progenitors by flow cytometry.

Patients and Methods

Patients Studied Bone marrow (BM) aspirates of 21 MDS patients, 13 men and

8 women with a median age of 66 years (range 62–77), were drawn into heparinized tubes. According to WHO classification, 6 had refractory anemia, 13 refractory cytopenia with multilineage dys-

plasia and 2 refractory anemia with excess blasts-1. All patients were diagnosed as having MDS on the basis of morphologic and cytogenetic criteria. Samples were taken prior to either cytotoxic or growth factor treatment. The control group consisted of 6 age-matched individuals all of whom had undergone BM aspiration for NMD (3 with idiopathic thrombocytopenic purpura, 3 with fever of undetermined origin proved to have nonhematologic dis-eases).

Agent R115777 (tipifarnib, Zarnestra) was provided by Johnson and

Johnson Pharmaceutical Research and Development as a white, crystalline powder. The drug was reconstituted in DMSO to a concentration of 1 m M (stock solution). Further dilutions were made in DMSO and concentrations of 2.5, 10, 25 and 50 n M were used for the experiments.

Progenitor Cell Isolation Mononuclear BM cells, isolated after density centrifugation,

were enriched for CD34+ cells with positive selection by using immunomagnetic beads according to the manufacturer’s instruc-tions (MACS, Miltenyi Biotec). The final purity of CD34+ cells was always above 90%, as measured by flow cytometry ( fig. 2 c).

Human BM Hematopoietic Clonogenic Assay in vitro MACS-selected CD34+ cells were plated in Methocult 4435

‘complete’ 1% methylcellulose medium, supplemented with 30% fetal bovine serum, 1% bovine serum albumin, 3 U/ml recombi-nant human (rh) erythropoietin, 10 –4 M 2-mercaptoethanol, 2 m M L -glutamine and the following cytokines: 50 ng/ml rh stem cell factor, 20 ng/ml rh GM-CSF, 20 ng/ml rh IL-3, 20 ng/ml rh IL-6 and 20 ng/ml rh G-CSF (Stem Cell Technologies). DMSO or tipi-farnib at the aforementioned concentrations was added at day 1. All cultures were performed in duplicates and the numbers of colonies were scored after 14 days of incubation at 37 ° C in a hu-midified incubator containing 5% CO 2 .

Apoptosis Assays MACS-selected CD34+ cells were plated at 2 ! 10 5 cells per

well in 48-well plates and cultured in RPMI supplemented with 10% FCS, 1% L -glutamine, 1% penicillin and streptomycin and 1 m M Hepes, in the presence of either DMSO or various concen-trations of tipifarnib. After 48 and 72 h cells were stained with CD34 (BD Biosciences), CD38-APC (BD Biosciences) and an-nexin V-FITC (BD Biosciences). The percentage of apoptotic, annexin V-positive, mature (CD34+CD38+) and immature (CD34+CD38–) progenitor cells was assessed by flow cytometry. Data acquisition and analysis was performed on a FACSCalibur, equipped with CellQuest Pro software (BD Biosciences). In a sec-ond set of experiments, purified CD34+ cells cultured as above for 24 h were stained with DiOC3 (Sigma-Aldrich) in order to detect the loss of mitochondrial transmembrane potential.

Statistical Analysis All analyses were performed using SPSS 13.0 software (SPSS

Science, Chicago, Ill., USA). Data are presented as mean 8 stan-dard error of the mean (SEM). The significance of the differences was assessed by paired or unpaired Student’s t test as appropriate. IC 50 values, i.e. the concentration of R115777 that inhibits colony formation to 50%, were determined by linear interpolation.

Tipifarnib in MDS Acta Haematol 2008;120:51–56 53

Results

Effect of Tipifarnib on the Clonogenic Potential of MDS and Normal Progenitors As shown in figure 1 , tipifarnib inhibited the colony

growth of total committed MDS progenitors at concen-trations of 2.5 n M (p = 0.017) and higher, in contrast to normal, nonclonal, CD34+ progenitors which were sensi-tive only at concentrations of 25 n M and above. The effect was more prominent in the MDS CFU-GM which sig-nificantly reduced their plating efficiency at 2.5 n M (p = 0.018), whereas erythroid and CFU-GEMM progenitors exhibited significant inhibition at 10 n M (p = 0.01) and above. Furthermore, indicative of the augmented suscep-tibility of the MDS CD34+ cells was the statistically sig-nificantly lower IC 50 value for MDS (23.5 n M ) compared to the normal progenitors (34.5 n M , p = 0.03).

Induction of Apoptosis after Incubation with Tipifarnib Early CD34+CD38– and mature CD34+CD38+ MDS

progenitors displayed equivalent annexin V binding in the presence of either DMSO or various concentrations of tipifarnib. Also, early apoptosis was not affected by the incubation period, as the apoptotic indexes were similar at 48 and 72 h ( fig. 2 a, b). Identical results were observed in both early and mature normal progenitors, on which tipifarnib did not induce significant apoptosis at the same time points ( fig. 3 a–d), indicating that, at least in vitro, early apoptosis on hematopoietic progenitors is not trig-gered by tipifarnib. In agreement with the annexin V staining, the mitochondrial potential � � m depolariza-tion of purified MDS progenitors was comparable when they were treated with either DMSO or tipifarnib at the indicated concentrations ( fig. 4 ).

Discussion

By unraveling the molecular mechanisms that govern leukemogenesis modern biology has identified several disease-related pathways that can be targeted by a variety of pharmaceutical agents. Tipifarnib, a potent and spe-cific inhibitor of FTase elicited significant responses in phase I and II trials in patients with advanced cancer and MDS. However, this clinical activity was unrelated to the degree of enzyme inhibition, whereas dose-limiting tox-icity due exclusively to myelosuppression was observed, both implying other unknown, potentially nonspecific, mechanisms of action [12–15] .

To our knowledge, a direct comparison of tipifarnib action in purified normal and MDS progenitors has not been conducted before. In the present study, we tested the effect of tipifarnib on the clonogenic capacity and apo-ptosis induction on selected CD34+ progenitors from MDS patients and patients with NMD. Since we used pu-rified CD34+ populations, NMD progenitors served as controls as they are not clonal and there is no evidence of an intrinsic defect of hematopoietic progenitors in pa-tients with the aforementioned diseases.

In the first part of our work, we showed that MDS pro-genitors are more sensitive in vitro to tipifarnib than their

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Fig. 1. MDS progenitors display increased in vitro sensitivity to tipifarnib. Selected CD34+ cells from patients with MDS ( a , n = 16) or NMD ( b , n = 6) were plated for short-term assays in cyto-kine replete medium. Inhibition of colony formation was signifi-cantly higher in the former, especially for the CFU-GM subset. Similar results were observed for the total CFU capacity ( c ). Val-ues are shown as mean and SEM, normalized to cultures grown only with DMSO (colony growth corresponding to 100%). The comparisons are made between DMSO and the various tipifarnib concentrations ( a , b ) or between MDS and NMD patients ( c ). * p ! 0.05 by paired Student’s t test in a and b or unpaired in c .

Kotsianidis et al. Acta Haematol 2008;120:51–5654

normal counterparts. The differences were statistically significant at concentrations up to 10 n M with MDS CFU-GM being the most sensitive, whereas adding tipifarnib at 25 n M and above generated the same effects in both groups. Similar results have been shown by Korycka et al. [16] who found significant differences between normal CFU-GM and AML colonies in various tipifarnib concentrations, whereas Liesveld et al. [17] also observed inhibition of CFU formation at doses between 10 and 100 n M . How-ever, the first author reports an IC 50 of 121.9 n M for nor-mal and 67.1 n M for leukemic progenitors which are both much higher than the IC 50 values of our data (23 and 34.5 n M for MDS and normal CD34+ cells, respectively), whereas in another study the IC 50 in normal progenitors was 46 n M [18] . Potential reasons for these discrepancies may include the different cell populations, i.e. AML versus MDS CD34+ cells, culture medium and cell purification methods that were used in the two studies. It is noticeable that the above authors used light density mononuclear BM cells, while in our culture experiments only purified BM progenitors were used in order to avoid any possible inter-actions between the various cell subsets in the mononu-

clear cell fraction (i.e. T cells and monocytes) that may influence the in vitro colony formation [19–22] .

Although data from in vitro systems are not always representative of the in vivo conditions because of the enormous biological variability of the latter, results from our study and others are in line with the effects of tipi-farnib in the clinical context. Phase II trials in MDS pa-tients support the use of 300 mg PO b.i.d. regimen (21 days in 4-week cycles), as it combines clinical efficacy with improved patient tolerance. Nevertheless, the de-gree of myelosuppression is still considerable, particular-ly drug-related grade 3–4 thrombocytopenia, which was consistently more frequent than neutropenia [13, 15] . In-terestingly, normal megakaryocytic progenitors are much more sensitive to tipifarnib (IC 50 : 9.1 n M ) [18] from both myelofibrosis nonmegakaryocytic progenitors (34 n M ) and – though not directly comparable – MDS progenitors in our study (23 n M ), thus substantiating the higher in vivo sensitivity of the megakaryocytic lineage in tipifarn-ib and indicating that even the optimum effective dose in MDS will probably result in some degree of thrombocy-topenia.

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Fig. 2. Measurement of annexin V expres-sion on MDS progenitor subsets. MDS CD34+ progenitors were cultured at the indicated concentrations of tipifarnib for 48 or 72 h. The percentage of annexin V-positive apoptotic cells in mature CD34+ CD38+ ( a ) or early CD34+CD38– ( b ) my-eloid progenitors was measured by flow cytometry and normalized to cultures grown only with DMSO (apoptotic index corresponding to 100%). The gating strat-egy is illustrated in c . No significant differ-ences in apoptosis were found in either early or mature progenitors. Values are shown as mean and SEM.

Tipifarnib in MDS Acta Haematol 2008;120:51–56 55

Beyond interfering with the Ras pathway, the mecha-nism of action of tipifarnib potentially involves inhibi-tion of a series of proteins other than Ras responsible for regulation of cell proliferation, apoptosis and migration [23] . However, induction of apoptosis by tipifarnib seems to depend on the experimental system that is used [24–26] , resulting in literature controversies, though most data converge to a secondary role for apoptosis as a major mechanism of tipifarnib action [10, 27] . In accord with these reports, we were not able to track any significant differences in apoptosis in both MDS and normal CD34+ progenitors in tipifarnib concentrations up to 25 n M . Ko-rycka et al. [16] comment that for concentrations below 100 n M extended incubation for more than 48 h is needed to demonstrate a significant increase in apoptosis, where-as Raponi et al. [28] found delayed onset of apoptosis in AML cell lines treated with 100 n M of tipifarnib. As shown in our results, there was no difference in early apoptosis either after 48 or 72 h, as demonstrated by two assays that detect distinct apoptotic events, whereas us-ing tipifarnib at 50 n M compromised cell viability (data not shown) making the interpretation of the results im-possible. Consistent with our data, Liesveld et al. [17] did not observe either late or early apoptosis in leukemic blasts cultured for 24 and 48 h in similar conditions. Ad-ditionally, the particularly low tipifarnib concentration

required for the inhibition of colony formation in MDS patients further suggests an effect on cell proliferation rather than apoptosis induction.

In conclusion, our results indicate that committed MDS progenitors and particularly CFU-GM display an up to 10-fold increase in sensitivity to tipifarnib com-pared to normal ones, whereas early apoptosis does not seem to be the mechanism responsible for this effect.

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Fig. 3. Tipifarnib does not induce significant apoptosis in either normal or MDS progenitors. Assessment of apoptosis by flow cytometry in purified mature ( a , c ) and early ( b , d ) myeloid progenitors from patients with MDS (n = 13) or NMD (n = 6). Similar percentages of annexin V expression were found after 48 h ( a , b ) and72 h ( c , d ) in both MDS and normal progenitors. Values are shown as mean and SEM and the data are normal-ized to cultures grown with DMSO only.

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Fig. 4. Measurement of DiOC3 expression on MDS progenitors. MDS CD34+ progenitors were cultured at the indicated concen-trations of tipifarnib for 24 h. The reduction of mitochondrial transmembrane potential was quantified by flow-cytometric analysis of DiOC3 uptake. Values are shown as mean and SEM and the data are normalized to cultures grown with DMSO only.

Kotsianidis et al. Acta Haematol 2008;120:51–5656

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These data, in combination with the results of previous studies which have demonstrated that tipifarnib also acts selectively against AML progenitors, provide further support for its clinical use in myeloid malignancies. How-ever, although the increased sensitivity of MDS progeni-tors allows for dose escalation, in vitro inhibition of nor-

mal and MDS progenitors may still occur at overlapping tipifarnib concentrations, suggesting that there is space for dose reduction in order to further alleviate the un-wanted myelosuppression as was recently shown in a phase I trial in MDS patients [29] .