Blood Journal
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FLT3-ITD–, but not BCR/ABL-transformed cells require concurrent Akt/mTor blockage to undergo apoptosis after histone deacetylase inhibitor treatment

  1. Dali Cai,
  2. Ying Wang,
  3. Oliver G. Ottmann,
  4. Peter J. Barth,
  5. Andreas Neubauer, and
  6. Andreas Burchert
  1. From the Universitätsklinikum Marburg und Gießen, Standort Marburg, Klinik für Hämatologie, Onkologie, und Immunologie, and Institut für Pathologie, Marburg; and University Hospital Frankfurt, Medizinische Klinik III, Frankfurt, Germany.

Abstract

Leukemias are differentially sensitive to histone deacytelase inhibitor (HDI)–induced apoptosis, but molecular reasons for this remain unclear. We here show that BCR/ABL-, but not FMS-like tyrosine kinase 3 (FLT3)–internal tandem duplication (ITD)–transformed 32D cells or primary acute myeloid leukemia (AML) blasts undergo apoptosis after treatment with the HDI valproic acid (VPA) plus all-trans retinoic acid (VPA/ATRA). A particular VPA/ATRA responsiveness of Philadelphia chromosome–positive (Ph+) acute lymphatic leukemia (ALL) was confirmed in a therapy-refractory patient in vivo. HDI-stimulated apoptosis in Ph+ cells was caspase dependent, but independent from Akt pathway inhibition. Conversely, separate blockage of the Akt/mTor-signaling pathway was a prerequisite for overcoming apoptosis resistance to VPA/ATRA in FLT3-ITD cells, and primary AML blasts (n = 9). In conclusion, constitutive Akt activation causes apoptosis resistance to VPA/ATRA in AML, but not in Ph+ leukemia. This warrants the application of HDI-based therapies in poor-risk Ph+ ALL, and the use of Akt/mTor inhibitors to overcome HDI resistance in AML.

Introduction

The BCR/ABL chromosomal translocation (Ph+) is detected in up to 30% of adult acute lymphoblastic leukemia (ALL) patients and associated with a detrimental prognosis.1,2 The FMS-like tyrosine kinase 3 (FLT3)–internal tandem duplication (ITD) oncogene has been identified as a frequent aberration in acute myeloid leukemia (AML).3 Both the FLT3-ITD and the BCR/ABL oncogenes cause malignant transformation and confer factor-independent growth (transformation) when expressed in the interleukin-3 (IL-3)–dependent cell line 32D.4,5 Specific BCR/ABL inhibition with imatinib mesylate (IM, Gleevec; Novartis, Basel, Switzerland) antagonizes transformation and causes impressive clinical remissions;6,7 however, IM resistance inevitably occurs in Ph+ ALL,8 and the overall prognosis in this disease remains poor even after allogenic transplantation.9 Histone deacetylase inhibitors (HDIs) block tumor growth via pleiotropic mechanisms that are only partially understood.10,11 Valproic acid (VPA) is a well-known anticonvulsant drug with HDI activity. VPA alone or in combination with all-trans retinoic acid (ATRA) has been reported to inhibit tumor growth and trigger differentiation12,13 and apoptosis11,14 of AML cells. However, in vivo, we and others recently demonstrated that the efficacy of VPA/ATRA in inducing remissions in myelodysplastic syndromes/AML is rather poor, whereas disease stabilization may be obtained more frequently.15-17 In BCR/ABL-positive cell lines, HDI have been shown to overcome IM resistance, particularly when combined with other inhibitors classes.18 Here we investigated determinants for the heterogenous efficacy of HDI in inducing apoptosis in acute leukemias.

Study design

Reagents

ATRA, VPA, z-VAD-fmk, and rapamycin were purchased from Sigma (Steinheim, Germany); SH6 was obtained from Alexis (Grünberg, Germany). The final concentrations of the different compounds used in this study were as follows: ATRA at 1 μM, VPA at 1 mM, SH6 at 10 μM, rapamycin at 10 nM, and caspase inhibitor z-VAD-fmk at 100 μMor200 μM.

Patients

Peripheral blood and bone marrow aspirates were obtained during routine punctures after obtaining written informed consent. Sample acquisition was approved by the local ethics committee of the University Marburg. In total, specimens of 9 patients with Ph+ ALL and 9 consecutive patients with AML were analyzed. For patient details, see Table S1, available on the Blood website (see the Supplemental Table link at the top of the online article). Mononuclear cells were isolated using Ficoll-Hypaque density gradient. The percentage of blasts was more than 80%. Fresh leukemic cells were cultured in RPMI 1640 supplemented with 30% fetal calf serum (FCS), 5 ng/mL IL-3 (Peprotech, Rocky Hill, NJ), 10 ng/mL IL-6 (Peprotech), and 20 ng/mL stem cell factor (SCF; Peprotech).

Assessment of apoptosis and cell cycle

For assessment of apoptosis, the Annexin V–FITC Apoptosis detection kit (Sigma-Aldrich, Steinheim, Germany) was used essentially as recommended by the manufacturer and as previously described.19 Cell cycle was analyzed by flow cytometry as previously described.20

Immunoblotting

Western blotting was performed as previously described19 using the following antibodies for detection of p21 (sc-6246), p27 (sc-528), and p45skp2 (sc-7164) (all from Santa Cruz Biotechnology, Santa Cruz, CA), and total Akt, pAKT (Ser 473), total p70S6 kinase, pp70S6 kinase (Thr 389), acetyl-histone H3 (lys9), and acetyl-histone H4 (lys8) from Cell Signaling Technology (Danvers, MA).

Stastistical analysis

Analysis of the statistical significance of a difference between the apoptosis induction frequency in different samples was done with the Mann Whitney U test. Differences were considered significant with P levels less than .05.

Results and discussion

32D cells were transformed by BCR/ABL and FLT3-ITD, resulting in IL-3–independent growth, respectively (Figure 1A). VPA/ATRA synergistically inhibited factor-independent growth in transformed lines and mediated a G0/G1 arrest (Figure 1A-B). VPA or VPA/ATRA blocked deacetylation of acetyl-histone H3 and H4 and up-regulated 2 known targets of HDI, p21waf1 and p27kip1 (Figure 1C).10 The level of accumulation of p27kip1 correlated with the degree of down-regulation of its specific ubiquitin ligase p45SKP2 21 and with growth arrest (Figure 1B-C). This suggested that VPA/ATRA synergistically inhibited proliferation via induction of p21waf1 and p27kip1. Notably, VPA/ATRA-induced growth arrest was comparable in BCR/ABL- and FLT3-ITD–transformed cell lines (Figure 1A-B), but apoptosis occurred exclusively in BCR/ABL-transformed 32D cells (Figure 1B, D). Likewise, BCR/ABL-transformed BaF3 pre-B cells, and BCR/ABL-positive K562 cells were susceptible to undergo VPA/ATRA-stimulated apoptosis (data not shown). This implied that BCR/ABL, but not the AML oncogene FLT3-ITD, confers a particular responsiveness of transformed cells to VPA/ATRA-induced apoptosis. Since Akt/mTor signaling provides important survival impulses for AML blasts,22 we investigated the Akt activation status before and after HDI treatment. Unexpectedly, VPA/ATRAdid not cause inhibition, but rather increased activation (phosphorylation) of Akt and p70S6 kinase in FLT3-ITD–and BCR/ABL-transformed cells (Figure 1E). Consequently, VPA/ATRA-induced cell death in BCR/ABL-positive cells occurred independently from Akt activation levels. In contrast, Akt could have contributed to apoptosis resistance against HDI in FLT3-ITD cells. Indeed, when Akt was specifically blocked in FLT3-ITD cells by SH6,23 as demonstrated by means of reduction of the phospho-Akt and phospho-p70S6K levels (Figure 1E), apoptosis resistance to VPA/ATRA was overcome (Figure 1D-E). Therefore, Akt blockage was required for apoptosis induction by VPA/ATRA in FLT3-ITD–positive cells, but not in BCR/ABL-expressing cells (Figure 1D-E). Furthermore, we found that HDI-mediated apoptosis in BCR/ABL-positive 32D cells and Ph+ primary cells was strictly caspase dependent because it could be blocked almost entirely by pan-caspase inhibition using z-VAD-fmk (Figure 2A). In contrast, VPA/ATRA/SH6-induced apoptosis in FLT3-ITD cells was not influenced by caspase inhibition (not shown).

Figure 1.

Differential effects of VPA/ATRA on cell cycle and apoptosis in BCR/ABL- and FLT3-ITD–transformed cells. (A) VPA, ATRA, and VPA/ATRA (A+V)–mediated proliferation inhibition in BCR/ABL (BA)–and FLT3-ITD–transformed 32D cells. Values indicate mean ± SD at each time point of 3 different experiments. (B) Cell-cycle distribution of BA and FLT3-ITD cells after treatment with indicated compounds for 72 hours. (C) Western blot analysis of protein lysates harvested after 48 hours of treatment with indicated compounds. Actin and total histone served as loading control. (D) Measurement of apoptosis using Annexin-FITC/propidium iodide staining after cells had been treated with VPA (V), ATRA (A), VPA/ATRA (A+V), or VPA/ATRA/SH-6 (A+V+SH-6) for 96 hours. Values represent mean ± SD of 3 independent experiments. Statistical significance of differences between the percentage of apoptosis after no treatment (co) and treatment with the respective compounds were assessed by Mann-Whitney U-test as indicated (*P < .01; **P < .001). (E) Western blotting of 30 μg protein lysates of 32D-BA and 32D-FLT3-ITD cells obtained after treatment with indicated compounds for 48 hours.

In order to validate our findings on primary cells, BCR/ABL-positive ALL blast samples (n = 9) and BCR/ABL-negative AML (n = 9) (Table S1) with greater 80% blasts in the peripheral blood were compared for HDI-stimulated apoptosis. In line with our hypothesis, VPA/ATRA elicited considerably more apoptosis in BCR/ABL-positive acute leukemias than in AML (P < .01; Figure 2B). In addition, as seen in the AML model cell line 32D-FLT3-ITD, resistance of primary AML blasts to undergo apoptosis after VPA/ATRA treatment was overcome by blocking Akt signaling (P < .03; Figure 2C). To inhibit the Akt pathway, the clinically available inhibitor of mTor, rapamycin (Rap), was used. Neither Rap nor VPA/ATRA alone was capable of eliciting apoptosis (Figure 2C). As shown in 2 patients with AML (no. 15 and no. 11), only the combination of VPA/ATRA and Rap, but neither drug alone, antagonizedAkt and p70S6K activity, which resulted in apoptosis (Figure 2D). This underscored the importance of Akt signal inhibition as a prerequisite for HDI-induced apoptosis in primary AML.

Figure 2.

VPA/ATRA treatment efficacy in primary leukemia cells in vitro and in vivo. (A) VPA/ATRA-induced apoptosis in BCR/ABL positive leukemia is caspase-dependent. 32D-BCR/ABL cells (top row) and a Ph+-ALL patient sample no. 9 (bottom row) were treated with VPA/ATRA (A+V), VPA/ATRA plus the pan-caspase inhibitor z-VAD-fmk, (A+V+ z-VAD-fmk), or mock-treated (control). Forty-eight hours after start of exposure to these compounds, apoptosis was assessed using FITC-Annexin/propidium iodide staining and fluoresecence activated cell-scanning (FACS) analysis. The total percentage of apoptotic cells is shown in the right quadrants, respectively. A representative experiment of at least 3 independent experiments for the 32D-BCR/ABL cell line and the patient sample is shown. Box-and-whisker plots for the comparison of VPA/ATRA (A+V)–mediated apoptosis in Ph+ ALL versus AML blasts in vitro (B) and the effect of VPA/ATRA (A+V), rapamycin (Rapa), or both (A+V+rapa) on apoptosis induction in primary AML (C). Lines in boxes indicate the median; boxes display data points located in the middle 2 quartiles of all data points. Whiskers extend to the 2 extreme values of all data points. Significant differences of medians of apoptosis induction, as determined by Mann-Whitney U-test, are indicated (**P < .01; *P < .03). (D) Western blotting of protein lysates generated from representative AML samples (patients no. 15 and no. 11) after in vitro treatment with indicated compounds for 48 hours. (E) A Ph+ ALL patient was treated with VPA/ATRA. Changes in peripheral white blood counts, percentage of granulocytes in the peripheral blood, and platelets are depicted during the first 45 days of treatment (i). Hypercellular bone marrow aspiration showing a homogenous ALL-blast infiltrate before treatment (ii, left panel), and bone marrow histology obtained after induction of remission with VPA/ATRA (ii, middle and right panels). Note the rare infiltration with CD34+/TdT+ ALL blasts in a hypocellular marrow (arrows in ii, middle and right panels).

An in vivo proof for the concept that Ph+ ALLs are particularly susceptible to HDI-induced apoptosis was observed in a patient who had failed all previous chemotherapeutic therapies, including IM. In this patient a combination treatment of oral VPA (increased to 300 mg twice daily) and ATRA (40 mg daily) was commenced. After 1 month of therapy, platelet and neutrophil blood counts had improved to almost normal values, and he recovered from a febrile pneumonia (Figure 2Ei). Whereas a bone marrow aspiration documented a hypercellular marrow with nearly 100% blastic infiltration before treatment (Figure 2Eii, left panel), the infiltration decreased to 20% to 30% of CD34+ and TdT+ blasts under VPA/ATRA therapy (Figure 2Eii, middle and right panels).

Together, Akt differentially dictates sensitivity to VPA/ATRA-mediated apoptosis in different types of acute leukemia. Whereas BCR/ABL-positive leukemias die after VPA/ATRA stimulation, FLT3-ITD–transformed cells and primary AML blasts require an independent Akt pathway inhibition to undergo HDI-stimulated apoptosis. These data warrant the clinical application of HDI in poor prognosis, Ph+ ALL. In AML, Akt/mTor signaling blocker may contribute to overcome HDI resistance.

Acknowledgments

The authors wish to thank Christine Barett for excellent technical assistance and Dr Hubert Serve for providing the 32D-FLT3-ITD cell line.

Footnotes

  • Reprints:

    Andreas Burchert, Klinikum der Philipps Universität Marburg, Klinik für Hämatologie, Onkologie, und Immunologie, Baldinger Straße, 35043 Marburg, Germany; e-mail: burchert{at}mailer.uni-marburg.de.
  • Prepublished online as Blood First Edition Paper, November 22, 2005; DOI 10.1182/blood-2005-08-3317.

  • Supported by the Deutsche José Carreras Leukämie-Stiftung e.V. R04/22f (A.B.) and R99/13 (A.B. and A.N.), by the P. E. Kempkes Stiftung (A.B.), by the Deutsche Forschungsgemeinschaft, SFB, Transregio 17 (A.B. and A.N.), and by a grant from the German Ministry of Education and Research (BMBF), Kompetenznetz “Akute und chronische Leukämien”–01 GI9980/6 (A.N).

  • The online version of this article contains a data supplement.

  • The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

  • Submitted August 16, 2005.
  • Accepted October 25, 2005.

References

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