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Keywords:

  • FLT3-ITD;
  • resistance;
  • point mutation;
  • acute myeloid leukaemia;
  • targeted therapy

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Fms-like tyrosine kinase (FLT3) mutations are the most frequent mutations in patients with acute myeloid leukaemia (AML) that confer a poor prognosis. Constitutively active FLT3-ITD (internal tandem duplications) mutations define a promising target for therapeutic approaches using small molecule inhibitors. However, several point mutations of the FLT3 tyrosine kinase domain (FLT3-TKD) have been identified to mediate resistance towards FLT3 tyrosine kinase inhibitors (FLT3-TKI), including secondary mutations of FLT3. We investigated the cellular effects of the recently characterised FLT3-TKI ponatinib (AP24534) on murine myeloid cells transfected with FLT3-ITD with or without additional point mutations of the FLT3-TKD including the (so far) multi-resistant F691I mutation. Ponatinib effectively induced apoptosis not only in the parental FLT3-ITD cell line but also in all stably transfected subclones harbouring additional FLT3-TKD point mutations (N676D, F691I or G697R). These observations correlated with a strong inhibition of FLT3-ITD and its downstream targets STAT5, AKT and ERK1/2 upon ponatinib incubation, as determined by Western blotting. We conclude that ponatinib represents a promising FLT3-TKI that should be further investigated in clinical trials. The targeted therapy of FLT3-ITD-positive AML with ponatinib might be associated with a lower frequency of secondary resistance caused by acquired FLT3-TKD mutations.

During the last decade, an increasing number of distinct molecular aberrations have been identified in acute myeloid leukaemia (AML) (Fröhling et al, 2005; Marcucci et al, 2011). Fms-like tyrosine kinase (FLT3) mutations are the most frequent kind of mutation in AML that is associated with an unfavourable outcome (Nakao et al, 1996; Yokota et al, 1997; Kottaridis et al, 2001; Yamamoto et al, 2001; Schnittger et al, 2002; Thiede et al, 2002). FLT3 internal tandem duplications (FLT3-ITD) and point mutations within the tyrosine kinase domain (TKD) represent the two main subtypes of FLT3 mutations. While FLT3-ITD mutations can be detected in about 25% of all AML patients, FLT3-TKD mutations are found in up to 10% of AML (Kottaridis et al, 2001; Yamamoto et al, 2001). All these FLT3 mutations confer a ligand-independent autophosphorylation of FLT3 that leads to the constitutive activation of STAT5 and important downstream targets, such as ERK1/2 or AKT (Hayakawa et al, 2000; Mizuki et al, 2000). The anti-apoptotic signalling of FLT3 mutations also affects the expression of Bcl-2 family members or survivin at the protein level (Minami et al, 2003; Brandts et al, 2005; Zhou et al, 2009). The up-regulation of Mcl-1 in a presumably STAT5-dependent manner is thought to be a key event in FLT3-induced leukemogenesis (Yoshimoto et al, 2009).

Distinct subclasses of FLT3 tyrosine kinase inhibitors (FLT3-TKI) have been developed, some of which are currently under investigation in prospective clinical trials (Kindler et al, 2010; Fathi & Levis, 2011; Pemmaraju et al, 2011).

Recent phase I/II clinical trials investigating FLT3-TKI monotherapy revealed a primary resistance in approximately 30% of FLT3-mutated AML patients (Stone et al, 2005; Zhang et al, 2008; Weisberg et al, 2009). Primary resistance is known to be either FLT3-independent, through the activation of alternative survival pathways, e.g. STAT5 and MAPK downstream signalling, or FLT3-dependent by mutations of FLT3 that are insensitive to the applied FLT3-TKI. Interestingly, the sensitivity towards FLT3-TKI was shown to diversify between different activating FLT3 mutations (Grundler et al, 2003; Chu & Small, 2009).

Nevertheless, the majority of patients treated with single compound FLT3-TKI experienced only transient response and the development of acquired secondary resistance reflects a special challenge in the treatment with FLT3-TKI (Kancha et al, 2007; Fischer et al, 2010).

One major mechanism of secondary TKI resistance is acquired genetic alterations within the FLT3-TKD. In particular, point mutations near the ATP binding site can cause secondary resistance that impairs drug binding to FLT3. Two in vivo acquired point mutations of FLT3 have been described in clinical investigations with FLT3-TKI. Treatment with midostaurin led to a secondary FLT3 mutation at codon 676 in one AML patient (Heidel et al, 2006). Furthermore, an A848 mutation of FLT3 caused secondary resistance in a patient with chronic myelomonocytic leukaemia (CMML) receiving treatment with sunitinib and sorafenib (von Bubnoff et al, 2010). This is similar to the mechanism of resistance to imatinib in chronic myeloid leukaemia (CML) that is potently overcome by the second line BCR/ABL1 inhibitors nilotinib or dasatinib (Gorre et al, 2001; Weisberg et al, 2007).

Contemporaneously, FLT3-TKD mutations at different positions, especially A627, N676, F691, and G697 were identified to confer varying degrees of resistance to several FLT3-TKI. Recently, in vitro analysis demonstrated a large number of FLT3 point mutations within the TKD1 and TKD2 with a differing sensitivity profile towards common FLT3 inhibitors, e.g. midostaurin (PKC412), sunitinib or sorafenib. Importantly, the F691I mutation was resistant to all compounds investigated (Cools et al, 2004; von Bubnoff et al, 2009). Interestingly, the Bis(1H-indol-2-yl)methanone c102 can partially overcome resistance of FLT3-ITD harbouring an additional point mutation at position N676 in Ba/F3 cells (Mahboobi et al, 2006; Heidel et al, 2009).

A recent pre-clinical evaluation (O'Hare et al, 2007) revealed ponatinib (AP24534, Ariad Pharmaceuticals, Cambridge, MA, USA) as a novel multi-targeted TKI. High-level resistance to imatinib, nilotinib, and dasatinib in the treatment of CML due to the BCR/ABL1T315I gatekeeper mutation is caused by an impaired inhibitor binding (O'Hare et al, 2007). Importantly, recent pharmacological research identified ponatinib as an orally active inhibitor of BCR/ABL1 including BCR/ABL1T315I mutation (O'Hare et al, 2009). Furthermore, ponatinib has shown inhibitory activity against tyrosine kinases that are involved in the pathogenesis of other haematological malignancies, including FLT3. A recent evaluation of ponatinib demonstrated a dose-dependent inhibition of FLT3 signalling and the induction of apoptosis in FLT3-ITD-positive AML cells including primary leukaemic blasts (Gozgit et al, 2011). It was also demonstrated that ponatinib effectively inhibited other receptor tyrosine kinases, especially all isoforms of the fibroblast growth factor receptor (Gozgit et al, 2012).

In this study, we sought to investigate the efficacy of ponatinib to inhibit FLT3-ITD harbouring additional point mutations within the FLT3-TKD that have been demonstrated to confer secondary resistance towards different classes of FLT3-TKI. In particular, we hypothesized that the multi-targeted inhibitor ponatinib might overcome resistance of the (so far) multi-refractory point mutation F691I of FLT3.

Materials and methods

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Cell lines, antibodies, and inhibitors

The human cell line MV4-11, which expresses FLT3-ITD while the corresponding wildtype allele is lost, was purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany). The four different Ba/F3 cell lines expressing either FLT3-ITD only (designated Ba/F3-ITD-598 due to a 36-basepair insertion corresponding to codon 598) or harbouring an additional point mutations (N676D, F691I, or G697R) were stably transfected as described previously and are further designated as Ba/F3-ITD-676, Ba/F3-ITD-691, and Ba/F3-ITD-697, respectively (Heidel et al, 2009). Cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 2 mmol/l l-glutamine and 1% of the antibiotics penicillin/streptomycin. The plasmids containing additional point mutations were kindly provided by Professor J. Duyster (Technical University, Munich, Germany).

The following antibodies were used for immunoblotting: anti-phospho-FLT3, anti-phospho-STAT5, anti-phospho-AKT, anti-phospho-ERK1/2, anti-STAT5, anti-AKT, anti-ERK1/2, anti-Mcl-1 (all from Cell Signaling Technology, Frankfurt, Germany). The anti-FLT3, the anti-Mcl-1 (flow cytometry) and the anti-β-Actin antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). The FLT3 inhibitor c102 was obtained from Professor Siavosh Mahboobi (University of Regensburg, Germany).

The FLT3 inhibitor sorafenib was purchased from Selleck Chemicals (Houston, TX, USA). Ponatinib was kindly provided by Ariad Pharmaceuticals. All inhibitors were dissolved in dimethyl sulphoxide (DMSO) 0·1% (v/v). The chemical structures of the used FLT3-TKI are shown in Fig S1.

Apoptosis assays

For Annexin V/propidium iodide (PI) apoptosis assay, cells were harvested and washed, after which 5 × 105 cells/ml per well were seeded in 24-well plates and different concentrations of FLT3 inhibitors were added to the wells while DMSO 0·1% (v/v) served as a negative control. The plates were incubated for 48 h at 37°C and then the samples were washed twice with cold phosphate-buffered saline (PBS). The cell pellet of each sample was resuspended in 200 μl staining solution containing 0·25 μl Annexin-V-allophycocyanin (APC), 0·5 μl PI, and 199 μl Annexin-V binding buffer and incubated for 15 min in the dark at room temperature. After addition of 400 μl Annexin-V binding buffer per sample, the cells were analysed by flow cytometry using a FACSCalibur (Becton Dickinson, Heidelberg, Germany).

Cell cycle analyses

For cell cycle analyses, cells were washed twice with PBS and resuspended in 1 ml PBS. Thereafter, 2 ml methanol were added per tube and incubated for 30 min on ice. Next, each cell pellet was resuspended in 500 μl PI/RNAse solution containing 450 μl PBS, 50 μl PI, and 5 μg (1 U) RNAse A. Following a 30-min incubation in the dark at room temperature, the samples were measured by flow cytometry.

Protein isolation and Western blot analysis

For protein analysis by Western blot, Ba/F3-ITD-598 and Ba/F3-ITD-691 cells were washed three times in serum-free medium and resuspended in serum-free medium at a density of 5 × 106 cells/ml. DMSO 0·1% (v/v) or different concentrations of sorafenib or ponatinib were added and incubated for 1 h at 37°C. In contrast, Mcl-1 expression of Ba/F3-ITD-598, Ba/F3-ITD-691 and MV4-11 cells was analysed after incubation of these cell lines with ponatinib for 24 h under standard conditions. Preparation of cell lysates was performed according to standard protocols using radioimmunoprecipitation assay buffer (RIPA) supplemented with fresh protease inhibitors and sodium-ortho-vanadate. Protein lysates were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a polyvinylidene fluoride (PVDF) membrane before blocking for 1 h. Next, the primary antibody was incubated overnight at 4°C. After the incubation with the secondary antibody, the membrane was visualised by adding enhanced chemiluminescence reagent. Digital images were taken using the Fujifilm LAS-3000 (Dusseldorf, Germany) and quantified by the AIDA Image Analyser.

Mcl-1 analyses by flow cytometry

The expression of Mcl-1 protein was detected by intracellular staining of Ba/F3 or MV4-11 cells and subsequent flow cytometric analysis. In detail, 1 × 106 cells/ml were seeded in six-well plates and incubated at varying concentrations of ponatinib or DMSO 0·1% (v/v) for 24 h at 37°C. Next, all samples were washed twice with medium before fixation was performed by adding 0·4 v/v of formalin (10%) per sample followed by a 10-min incubation. After this, the cells were centrifuged and resuspended in 5 ml cold methanol for permeabilization, followed by a 20-min incubation at 4°C and two washing steps each with 5 ml staining solution (PBS plus 1% bovine serum albumin). The cells were then resuspended in 200 μl staining solution and equally divided on two FACS tubes per sample (specimen and control). The anti-Mcl-1 primary antibody was added (only to specimen) and incubated for 30 min. All samples, including the controls, were resuspended in 100 μl staining solution containing the fluorochrome-coupled secondary antibody for 30 min in the dark. After repeated washes, the samples were resuspended in 400 μl staining solution for measurement on a FACSCalibur.

Statistics

The Student′s t-test was used for statistical analyses and P values < 0·05 were considered statistically significant. Unless otherwise indicated, all experiments were performed in triplicates.

Results

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Ponatinib induces apoptosis in FLT3-ITD-positive Ba/F3 cells harbouring additional point mutations

To evaluate the efficacy of ponatinib, we investigated the induction of apoptosis in all four Ba/F3 cell lines after 48 h of incubation with increasing concentrations of ponatinib, sorafenib, or c102 (Fig 1). The efficacy was determined by the 50% inhibitory concentration (IC50) as summarized in Table 1.

image

Figure 1. Analysis of apoptosis in Ba/F3 cell lines upon treatment with FLT3 inhibitors. Annexin/propidium Iodide assay was performed to examine apoptosis in murine Ba/F3 cell lines harbouring ITD without (598) or with resistance-mediating mutations (676, 691, and 697) dependent on the incubation (48 h) with c102, sorafenib or ponatinib by fluorescence-activated cell sorting (FACS). Dimethyl sulphoxide 0·1% (v/v) served as a negative control. Values represent the means ± SD of total Annexin V-positive cells from three independent experiments.

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Table 1. Overview of the cellular IC50 values for the induction of apoptosis in Ba/F3 cell lines upon treatment with different FLT3 inhibitors.
InhibitorIC50
Ba/F3-ITD-598Ba/F3-ITD-676Ba/F3-ITD-691Ba/F3-ITD-697
c1020·06 μmol/l1·2 μmol/l>5 μmol/l>5 μmol/l
Sorafenib4·7 nmol/l566 nmol/l>1000 nmol/l154 nmol/l
Ponatinib0·3 nmol/l43 nmol/l3·8 nmol/l77 nmol/l

Using Ba/F3-ITD-598 cells, c102 induced apoptosis with an IC50 of 0·06 μmol/l, and with an IC50 of 1·2 μmol/l in Ba/F3-ITD-676 cells while in Ba/F3-ITD-691 and Ba/F3-ITD-697 cells apoptosis was not significantly affected within a concentration range of c102 up to 5 μmol/l. The Ba/F3-ITD-598 cell line also showed an increasing percentage of apoptotic cells during treatment with sorafenib (IC50 = 4·7 nmol/l), whereas Ba/F3-ITD-697 cells were moderately resistant to sorafenib (IC50 = 154 nmol/l). Strong resistance of Ba/F3-ITD-676 (IC50 = 566 nmol/l) and Ba/F3-ITD-691 (IC50 > 1000 nmol/l) cells towards sorafenib was detected. In contrast, after incubation with ponatinib, the Ba/F3-ITD-598 and Ba/F3-ITD-691 cell lines showed a significant induction of apoptosis, indicated by an IC50 of 0·3 and 3·8 nmol/l, respectively. In Ba/F3-ITD-676 (IC50 = 43 nmol/l) and Ba/F3-ITD-697 (IC50 = 77 nmol/l) mutant harbouring cells, however, higher concentrations of ponatinib were necessary to induce apoptosis.

Inhibition of Ba/F3 cell proliferation by different FLT3 inhibitors

Next, we analysed the cell cycle distribution in 48 h inhibitor-treated Ba/F3 cell lines stained with PI by flow cytometry (Fig 2). In particular, the analysis was focused on the cell cycle S+G2/M fraction indicating the percentage of proliferating cells.

image

Figure 2. Cell cycle analysis of Ba/F3 cell lines harbouring FLT3-ITD, following incubation with FLT3 inhibitors. Murine Ba/F3-ITD-598 (A) or Ba/F3-ITD-691 (B) cell lines were incubated at different concentrations of c102, sorafenib, or ponatinib for 48 h. Cell cycle analysis was performed by fluorescence-activated cell sorting (FACS), while dimethyl sulphoxide (DMSO) 0·1% (v/v) served as a negative control. Mean values (percentages of sub-G1, G0/G1 and S+G2/M) of three experiments are indicated.

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As a consequence of treatment with c102, there was a significant concentration-dependent decrease of the S+G2/M fraction in Ba/F3-ITD-598 cells with an IC50 between 0·1 and 0·3 μmol/l. In Ba/F3-ITD-691 mutants a moderate influence of c102 on cell cycle was detectable at a concentration of 5 μmol/l. The incubation with sorafenib also resulted in a significant decrease in S+G2/M fraction of Ba/F3-ITD-598 cell cycle (IC50 = 10–30 nmol/l). In contrast, in Ba/F3-ITD-691 cells no inhibition of proliferation was detected up to a concentration of 100 nmol/l sorafenib.

Nevertheless, treatment of Ba/F3-ITD-598 cells with ponatinib resulted in a concentration-dependent inhibition of proliferation with an IC50 between 3 and 10 nmol/l. Importantly, in ponatinib-treated Ba/F3-ITD-691 mutants, a reduction of the S+G2/M fraction was achieved with an IC50 value of 10 nmol/l.

Cell cycle analysis of FLT3-ITD harbouring Ba/F3 cell lines Ba/F3-ITD-676 and Ba/F3-ITD-697 are shown in Fig S2. They were consistent with the apoptosis data in that ponatinib was more potent than sorafenib in inhibiting the proliferation of Ba/F3-ITD-676 cells and, to a lesser extent, Ba/F3-ITD-697 cells.

Ponatinib inhibits autophosphorylation and downstream signalling of FLT3-ITD

We next evaluated the cellular effects of the FLT3 inhibitors sorafenib and ponatinib on the phosphorylation status of FLT3-ITD and the phosphorylation of STAT5 in Ba/F3 cells. In detail, Ba/F3-ITD-598 and Ba/F3-691 cells were incubated with either sorafenib or ponatinib at different concentrations as indicated in Fig 3A and B, respectively. Ba/F3-ITD-598 cells showed an almost complete inhibition of FLT3-ITD autophosphorylation after incubation with sorafenib at a concentration of 10 nmol/l while ponatinib treatment with 30 nmol/l was necessary to achieve a comparable reduction of receptor phosphorylation. Interestingly, the concentration-dependent effects of both inhibitors on FLT3 phosphorylation in Ba/F3-ITD-691 cells were opposite to the effects in Ba/F3-ITD-598 cells. In detail, ponatinib significantly reduced the phosphorylation of F691I-mutated FLT3-ITD at 10 nmol/l, whereas a higher concentration of sorafenib (30 nmol/l) was required to inhibit FLT3 phosphorylation at the same level in Ba/F3-ITD-691 cells. There was a good correlation between the inhibition of FLT3 autophosphorylation and the detectable amount of phosphorylated STAT5 in both Ba/F3 cell lines.

image

Figure 3. The influence of sorafenib and ponatinib on the phosphorylation status of FLT3 and its downstream targets in Ba/F3 cells. Cells were treated with each of both compounds for 1 h and phosphorylation in cell lysates was analysed by immunoblotting using phosphospecific antibodies for FLT3, STAT5, AKT and ERK1/2. Equal loading was controlled by reprobing the blots with FLT3-, STAT5-, AKT- or ERK1/2- antibodies or β-actin, respectively.

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We were further interested to analyse the effects of sorafenib and ponatinib on such important signalling pathways as the PI3K-AKT and the MAPK pathway in Ba/F3-ITD-598 and Ba/F3-ITD-691 cells, respectively. We therefore determined the concentration-dependent influence of sorafenib and ponatinib on the phosphorylation of AKT and ERK1/2 in both Ba/F3 cell line models (Fig 3C and D, respectively). Treatment of Ba/F3-ITD-598 cells with sorafenib at a concentration of 30 nmol/l revealed a significant increase of AKT phosphorylation while there was no induction of ERK1/2 phosphorylation. Importantly, a similar phenomenon was observed after incubation of Ba/F3-ITD-691 cells with 10 nmol/l sorafenib. There was not only an increase of AKT phosphorylation but also an enhanced phosphorylation of ERK1/2.

Treatment of Ba/F3-ITD-598 or Ba/F3-ITD-691 cells with ponatinib at different concentrations did not demonstrate such a “paradox signalling” at the levels of AKT or ERK1/2 phosphorylation. Thus, the basal phosphorylation of AKT in Ba/F3-ITD-598 cells – as well as the phosphorylation of ERK1/2 in Ba/F3-ITD-691 cells – was markedly reduced at the lowest concentrations of ponatinib (3 nmol/l).

Expression of Mcl-1 in Ba/F3-ITD-598 and Ba/F3-ITD-691 cells

Mcl-1 expression was selectively examined in the Ba/F3-ITD-598 and Ba/F3-ITD-691 cell lines. In Ba/F3-ITD-598 cells (Fig 4A), flow cytometry analysis demonstrated a high expression of Mcl-1 in the control represented by the presence of DMSO only. Increased concentrations of ponatinib resulted in a significant and strong down-regulation of Mcl-1 expression. In detail, an inhibitor concentration ranging from 3 to 10 nmol/l caused a 50% reduction of Mcl-1 expression in Ba/F3-ITD-598 cells. Ponatinib concentrations higher than 30 nmol/l did not show any further influence on Mcl-1 expression and regulation. These observations correlate with immunoblot analyses of Mcl-1 expression using an antibody that recognizes both potentially expressed isoforms of Mcl-1 (Fig 4B). Importantly, only the anti-apoptotic isoform of Mcl-1 could be detected by Western blot in all investigated cell lines. In contrast to Ba/F3-ITD-598 cells, only a low Mcl-1 expression was detectable in Ba/F3-ITD-691 cells by flow cytometry and no significant regulation upon ponatinib treatment was seen (Fig 4C). Western blot analysis of Mcl-1 confirmed the low level and unaffected regulation of Mcl-1 expression after ponatinib treatment of Ba/F3-ITD-691 cells (Fig 4D).

image

Figure 4. Analysis of Mcl-1 expression in ponatinib-treated Ba/F3-ITD-598 (A) or Ba/F3-ITD-691 cells (B). Flow cytometry was used to analyse Mcl-1 expression of cells treated with ponatinib for 24 h and then stained with an anti-Mcl-1 antibody. Open histograms represent isotype controls, filled histograms show fluorescence intensities of Mcl-1, whereas the y-axes indicate mean fluorescence intensity of Mcl-1. DMSO 0·1% (v/v) served as a negative control. For Western blot analysis of Mcl-1 expression, cells were treated with ponatinib as described for flow cytometry analyses and cell lysates were prepared as described above.

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Discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We aimed to investigate the efficacy of the recently characterized FLT3-TKI, ponatinib, to induce apoptosis in FLT3-ITD-positive cells dependent on the presence of additional FLT3-TKD mutations that are known to cause resistance to other FLT3-TKI. We were especially interested to determine whether ponatinib could inhibit FLT3-ITD containing the multi-resistant F691I mutation.

The analyses of apoptosis induced by c102, sorafenib, or ponatinib demonstrated a strong resistance of the FLT3 G697R mutation to c102 and a strong resistance of the FLT3 F691I mutation towards c102 and sorafenib up to a concentration of 5 and 1 μmol/l, respectively. These data agree with previously published results (von Bubnoff et al, 2009; Heidel et al, 2009). Nevertheless, exposure of Ba/F3-ITD-691 cells to higher concentrations of sorafenib (up to 10 μmol/l) resulted in apoptosis of Ba/F3-ITD-691 cells with an IC50 value of approximately 3 μmol/l (data not shown).

Furthermore, in Ba/F3 cells expressing FLT3-ITD, apoptosis was induced at subnanomolar concentrations of ponatinib. For the induction of apoptosis in the parental interleukin 3-dependent Ba/F3 cell line, IC50 values of approximately 1800 nmol/l were determined demonstrating that no relevant off-target effect of ponatinib contributes to induction of apoptosis of FLT3-ITD-transfected Ba/F3 cells at low concentrations (O'Hare et al, 2009). Importantly, the FLT3-ITD protein remains highly sensitive to ponatinib in presence of a concomitant F691I point mutation. In addition, ponatinib also induced apoptosis when FLT3-ITD was associated either with the N676D or the G697R mutation. For both of these FLT3-TKD mutations, the IC50 values were, however, one log rank higher as compared with the F691I-mutated FLT3-ITD variant. In consideration of pharmacokinetic data demonstrating a steady state plasma level of more than 100 nmol/l ponatinib in humans, the F691I point mutation of FLT3-ITD should be very effectively inhibited in vivo, and the N676D and the G697R point mutation of FLT3 can also be assumed to be sensitive to ponatinib (Cortes et al, 2010).

For sorafenib, pharmacokinetic data demonstrated steady state plasma levels of about 5 μmol/l when the compound was orally administered at the maximum dosage level. It is therefore possible that sorafenib might also at least inhibit FLT3-ITD-positive leukaemia cells harbouring an additional F691I point mutation when applied at these high doses. Nonetheless, we hypothesize that sorafenib should not be equivalent to ponatinib concerning its efficacy to induce apoptosis of cells harbouring the FLT3-ITD-F691I because the ponatinib IC50 value of 3·8 nmol/l is far below the regular plasma level for this inhibitor, which is more than 10-fold of the IC50.

In addition, we performed cell cycle analyses of all cell lines investigated in this study dependent on the incubation with ponatinib. For Ba/F3-ITD-598 cells, an IC50 of between 3 and 10 nmol/l was demonstrated for the inhibition of proliferation based on the S+G2/M fraction. A similar efficiency of cell cycle inhibition could be detected in Ba/F3-ITD-691 cells. These data support the notion that ponatinib can effectively inhibit proliferation of both cell lines, as well as that of MV4-11 cells (Fig S3), at concentrations below 10 nmol/l and induce apoptosis in a similar concentration range as demonstrated by the Annexin V/PI assay.

Next, we investigated the inhibition of FLT3-ITD and its downstream signalling pathways (STAT5, AKT and ERK1/2) by the FLT3-TKIs sorafenib and ponatinib in Ba/F3-ITD-598 and Ba/F3-ITD-691 cells, respectively. The inhibition of FLT3 phosphorylation, as determined by phospho-specific immunoblot analyses of tyrosine 589, showed a good correlation with the dose-dependent inhibition of STAT5 activation for both FLT3-TKI and in both Ba/F3 cell lines. Thus, the IC50 value of phospho-STAT5 can be estimated at 3 nmol/l for ponatinib in both Ba/F3-ITD-598 and Ba/F3-ITD-691 cells. Furthermore, the IC50 value of sorafenib was significantly lower when STAT5 phosphorylation was analysed in Ba/F3-ITD-598 cells as compared with Ba/F3-ITD-691 cells (3 vs. 30 nmol/l). These data support the observation that the F691I mutation is resistant to the FLT3-TKI sorafenib. Importantly, a significant reduction of STAT5 phosphorylation was observed when Ba/F3-ITD-691 cells were incubated with sorafenib at a concentration of 30 nmol/l for 1 h. This seems to be contradictory to the results of the Annexin V/PI assay, wherein we confirmed the strong resistance of F691I-mutated FLT3-ITD to sorafenib in Ba/F3-ITD-691 cells (IC50 > 1000 nmol/l) (von Bubnoff et al, 2009). One possible explanation is based on pharmacokinetic aspects, i.e., that a short half-life of the inhibitor may cause a reduction of sorafenib concentration during the 48 h incubation.

We also investigated the impact of sorafenib and ponatinib on downstream pathways, e.g. the phosphorylation of AKT and ERK1/2, in Ba/F3-ITD-598 and Ba/F3-ITD-691 cells, respectively. Incubation of both murine cell lines with different concentrations of ponatinib resulted in a strong reduction of phospho-AKT in Ba/F3-ITD-598 cells and a significant decrease of phospho-ERK1/2 in Ba/F3-ITD-691 cells. In contrast, the basal AKT phosphorylation in Ba/F3-ITD-691 cells and the basal phosphorylation of ERK1/2 in Ba/F3-ITD-598 cells were both too low and therefore not appropriate to determine a concentration-dependent effect of the FLT3-TKI. Surprisingly, in both Ba/F3 cell lines, incubation with sorafenib was associated with an increase of AKT phosphorylation at one distinct concentration (30 nmol/l in Ba/F3-ITD-598 cells versus 10 nmol/l in Ba/F3-ITD-691 cells). Furthermore, the phosphorylation of ERK1/2 also increased when Ba/F3-ITD-691 cells were treated with 30 nmol/l sorafenib. Mechanisms of such a paradoxical downstream activation are a matter of speculation. Similar observations have been described for other multi-targeted inhibitors like the BCR/ABL1 inhibitor imatinib (Chu et al, 2004; König et al, 2008).

We further determined the expression of the important anti-apoptotic protein Mcl-1 by intracellular staining and subsequent analysis by flow cytometry. We demonstrated a strong reduction of Mcl-1 expression in Ba/F3-ITD-598 cells with increasing concentrations of ponatinib. Surprisingly, not only the inhibition but also the expression of Mcl-1 itself was much lower in Ba/F3-ITD-691 cells. Importantly, ponatinib at 10 nmol/l, which effectively induced apoptosis in F691I-mutated Ba/F3-ITD cells, demonstrated only a slight reduction of Mcl-1 expression. Because immunoblotting of phospho-STAT5 does not necessarily represent its activation state to induce downstream pathways, a potential explanation for the low Mcl-1 expression observed in Ba/F3-ITD-691 cells might be reflected by a discrepancy between STAT5 phosphorylation and its activation. In detail, F691I-specific induction of signalling pathways that can directly or indirectly inhibit STAT5-dependent transcriptional activity might modulate FLT3-ITD-induced Mcl-1 expression. Further experiments will be necessary to address these open questions.

Taken together, the development of resistance towards FLT3 inhibitors remains a serious challenge in the treatment of AML. The knowledge about the underlying molecular mechanisms in the pathogenesis of resistance is necessary to prevent them and further improve the therapeutic options for FLT3-ITD-positive AML patients. Ponatinib, however, is a new promising compound for overcoming secondary resistance towards available FLT3-TKI due to the evolution of leukaemic subclones acquiring an additional point mutation in the TKD of FLT3. Furthermore, ponatinib might also be associated with a lower mutation rate when applied as first line therapy with FLT3-TKI in AML. Therefore, clinical trials are warranted to elucidate the efficacy and in vivo resistance profile of ponatinib in FLT3-ITD-positive AML.

Acknowledgements

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This work was supported by a grant from the Deutsche Krebshilfe foundation (Az. 108868). The plasmids containing additional point mutations were kindly provided by Professor J. Duyster (Klinikum rechts der Isar, Technical University, Munich, Germany). The authors are grateful to Ariad Pharmaceuticals, Cambridge, MA, USA, for providing Ponatinib.

References

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
FilenameFormatSizeDescription
bjh9085-sup-FigS1.TIFimage/TIF136KFig S1. Chemical structure of the used FLT3 inhibitors compound 102 (A), sorafenib (B), and ponatinib (C).
bjh9085-sup-FigS2.TIFimage/TIF1742KFig S2. Cell cycle analysis of FLT3-ITD harbouring Ba/F3 cell lines after incubation with FLT3 inhibitors.
bjh9085-sup-FigS3.TIFimage/TIF2761KFig S3. Influence of FLT3-TKI on apoptosis, cell cycle and Mcl-1 expression in MV4-11 cells.

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