Analysis of FLT3-ITD mutations

For detection of FLT3-ITD, polymerase chain reaction (PCR) amplification of genomic DNA was carried out using primers 11F and 12R located on FLT3 exons 14 and 15 as previously described.³¹ PCR products were separated by electrophoresis using 3% Nusieve GTG agarose gel (Cambrex Bio Science Rockland) and cut out from the gel, purified with a QIAquick Gel Extraction kit (QIAGEN), and cloned into the PCR II-TOPO vector (Invitrogen) according to the manufacturer's recommendations. Sequencing was performed using the ABI PRISM BigDye Terminator V 1.1 Cycle Sequencing Kit and 21M13 and T7 primer on an ABI 310 Prism sequencer (Applied Biosystems).

Antibodies, cell staining, and sorting

CD34⁺ cells were enriched from BM cells using the Indirect CD34 MicroBead Kit (Miltenyi Biotec) as described previously.³² For analyses and sorting, cell samples were stained with cyanin 5–phycoerythrin (PE)– or phycoerythrin-Cy5–conjugated lineage mixture, including cyanin 5–PE– or phycoerythrin-Cy5–conjugated CD3 (HIT3a), CD4 (RPA-T4), CD8 (RPA-T8), CD10 (HI10a), CD20 (2H7), CD11b (ICFR44), CD14 (RMO13), GPA (GA-R2), fluorescein isothiocyanate–conjugated anti-CD90 (Thy-1) (5E10) or anti-CD45RA (HI100), PE-conjugated anti-CD117 (c-Kit) (YB5.B8) or anti-FLT3 (CD135) (4G8), anti-CD123 (interleukin-3Rα [IL-3Rα]) (6H6), allophycocyanin–conjugated anti-CD34 (8G12), and biotin-conjugated anti-CD38 (HIT2) antibodies. Streptavidin-conjugated allophycocyanin was used for visualization of the biotinylated antibodies (BD Pharmingen). Nonviable cells were excluded by propidium iodide (PI) staining. HSCs (CD34⁺CD38⁻CD90⁺Lin⁻ cells) and myeloid progenitors, including common myeloid progenitors (CD34⁺CD38⁺CD123⁺ CD45RA^loLin⁻), granulocyte-macrophage progenitors (GMPs: CD34⁺CD38⁺CD123⁺ CD45RA^loLin⁻), and megakaryocyte-erythroid progenitors (CD34⁺CD38⁺CD123⁻CD45RA⁻Lin⁻) were isolated from normal and AML BM cells by fluorescence-activated cell sorter (FACS, FACSAria; BD Biosciences).^32,33 The sorted cells were subjected to an additional round of sorting using the same gate to eliminate contaminating cells and doublets. HSCs and GMPs in the murine bone marrow were purified according to the method described previously.³⁴

Cell culture and apoptosis measurement

To test the effect of FLT3 stimulation on the leukemic cells, the sorted cells were prepared in the fetal calf serum (FCS)–free condition media with human (h) FLT3 ligand (FL, 20 ng/mL) or human stem cell factor (SCF, 20 ng/mL). After culture of 30 minutes, cells were subject to analysis of gene expression. Leukemic cell lines, including FLT3-ITD–positive cell lines, MV4-11, were cultured in RPMI-1640 medium containing 10% fetal calf serum (FCS) in the absence or presence of PKC-412 (LC Laboratories), Ly294002 (EMD Biosciences), AG490 (EMD Biosciences), or U0126 (Cell Signaling Technology). Cultured cell lines were picked up for analyses of changes in transcript and protein expression and apoptotic status. Apoptosis of cell lines treated with PKC-412 was evaluated using annexin V (BD Pharmingen) and PI staining. The living cells were defined as annexin V⁻/PI⁻ among the live-gated cells.

Quantitative real-time PCR assay

Total RNA was extracted from 2000 purified cells using Isogen reagent (Nippon Gene) according to the manufacturer's protocols. RNA was reverse transcribed to cDNA using a TaKaRa RNA PCR Kit (Takara). The mRNA levels were quantified in triplicate using a real-time PCR (7500 Real-Time PCR system; Applied Biosystems). β₂-Microglobulin mRNA was separately amplified in the same plate to be used for internal control. The primers and probes were designed by Primer Express software (Applied Biosystems) as follows: BCL-2 (the forward primer, 5-ctgggatgcctttgtggaa-3; the reverse primer, 5-cagccaggagaaatcaaacaga-3; and the probe, 5-FAM-tgtacggccccagcatgcgg-TAMRA-3), BCL-xL (the forward primer, 5-aatgaccacctagagccttgga-3; the reverse primer, 5-tgctgcattgttcccatagagt-3; and the probe, 5-FAM-cggcggctgggatacttttg-TAMRA-3), MCL-1 (the forward primer, 5-caaaacgggactggctagttaaa-3; the reverse primer, 5-ccttctaggtcctctacatggaaga-3; and the probe, 5-FAM-aaagaggctgggatgggtttgtggag-TAMRA-3), and β₂-microglobulin (the forward primer, 5-tgactttgtcacagcccaagata-3; the reverse primer, 5-aatgcggcatcttcaaacct-3; and the probe, 5-FAM-ttaagtgggatcgagacatgtaagcagcatc-TAMRA-3). Collected data were analyzed with Sequence Detector software (Applied Biosystems). All the samples and PCR analyses were carried out in duplicate, and negative controls were included in each run. Data were expressed as fold changes in gene expression relative to those from normal BM or normal HSCs.

Intracellular flow cytometry

Cells were fixed and permeabilized, and labeled with the rabbit polyclonal antibody MCL-1 (S-19; Santa Cruz Biotechnology), followed by the second staining with fluorescein isothiocyanate– or PE-conjugated antirabbit antibodies for visualization.

Western blot analysis

Cells (10⁹) were lysed as described.³⁵ Lysates were then denatured in an equal volume of 2× sodium dodecyl sulfate (SDS) sample buffer, resolved by a 10% SDS–polyacrylamide gel electrophoresis gel, and electrotransferred to nitrocellulose membranes in non–SDS-containing transfer buffer (25mM tris(hydroxymethyl)aminomethane, 0.2M glycine, 20% methanol, pH 8.5). Western blotting was performed with rabbit anti–human MCL-1 sera (1:5000; Sigma-Aldrich), anti–human phospho-Akt (Cell Signaling Technology), anti–human phospho-mitogen-activated protein kinase kinase 1/2 (MEK1/2; Cell Signaling Technology), or mouse anti–β-actin monoclonal antibody (1:5000; Sigma-Aldrich), followed by 1:15000 dilution of antirabbit or antimouse horseradish peroxidase–conjugated immunoglobulin G (Jackson ImmunoResearch Laboratories). Blots were developed with enhanced chemiluminescence plus kit (Amersham Biosciences). The chemiluminescence intensity was monitored by laser3000 (FujiFilm) instrument. We quantitated band intensity of the proteins using ImageGauge software (FujiFilm) and normalized their expression in reference to β-actin levels. Using these normalized data, relative expression (RE) is subsequently calculated as fold changes in protein expression compared with the controls.

Retroviral transduction of HSCs and progenitors

The constructs of FLT3-ITD N51and FLT3-ITD tyrosine to phenylalanine mutant at sites 589 and 591, which are occult STAT5 binding sites, were previously described.³⁰ The retrovirus vector was transfected into 293T cells with gag and pol expression plasmids by CaPO₄ coprecipitation. The supernatant from transfected cells was collected after 48 hours and immediately stored at −80°C until use. FACS-purified murine HSCs were prestimulated in RPMI-1640 medium containing 20% FCS in the presence of murine (m) SCF (20 ng/mL), murine leukemia inhibitory factor (10 ng/mL) and mFL (10 ng/mL) for 18 hours. Cells were subsequently plated onto recombinant fibronectin fragment-coated culture dishes (RetroNectin dish; Takara) with 1 mL of virus supernatant containing identical cytokine cocktail and cultured for 48 hours. For progenitor transduction, FACS-purified cells were cultured for 48 hours on RetroNectin dishes with 1 mL of virus supernatant containing mSCF and mIL-11 (10 ng/mL) for GMPs

The enforced expression and knockdown experiments for MCL-1

Human MCL-1 cDNA plasmid was purchased from Open Biosystems. The coding sequence of MCL-1 in this plasmid was amplified by PCR using primers containing additional EcoRI site (5′-cggaattcggcaatgtttggcctc-3′) and BamHI site (5′-gcggatcctcttattagatatgc-3′) and cloned into pEGFP-C3 vector (Clontech). To suppress the expression level of MCL-1, we generated a vector that possesses expression cassettes of short hairpin (sh) RNA against MCL-1 using the BLOCK-iT PolII miR RNAi Expression Kit (Invitrogen) according to the manufacturer's recommendation. Target sequences were selected using BLOCK-iT RNAi Designer (Invitrogen). Briefly, oligonucleotides were synthesized to form shRNA on transcription, in order of sense, loop (lower case), and antisense sequences as follows: MCL-1 top strand oligo, 5′-tgctgttgatgtccagtttccgaagcgttttggccactgactgacgcttcggactggacatcaa-3′; MCL-1 bottom strand oligo, 5′-cctgttgatgtccagtccgaagcgtcagtcagtggccaaaacgcttcggaaactggacatcaac-3′. These oligonucleotides were annealed with their complementary ones and cloned subsequently into pcDNA 6.2-GW/EmGFP-miR expression vector. After confirming their sequences, expression vectors were transduced into MV4-11 cell lines by Amaxa Nucleofector System (Lonza). Cells (2 × 10⁶) were suspended in 100 μL of Nucleofector solution with 2 μg of negative control vector or subcloned plasmid. Pulses were delivered with Nucleofector device using program Q-001 (Lonza).

STAT5 knockdown experiment

Double-stranded STAT5 siRNA was purchased from Invitrogen. STAT5 siRNA was transduced into MV4-11 cell lines by Amaxa Nucleofector System (Lonza). Cells (2 × 10⁶) were suspended in 100 μL of Nucleofector solution with 1.5 μg of siRNA. Pulses were delivered with Nucleofector device using program Q-001.

The presence of FLT3-ITD marks a fraction of AML with highest levels of MCL-1 expression

Figure 2 shows the real-time PCR analysis of MCL-1 mRNA levels in normal and leukemic BM mononuclear cells. AML BM cells expressed MCL-1 in all 30 cases at a level approximately 5-fold higher than that of normal BM cells, irrespective of their FAB types (Figure 2A). We have shown that, in normal human hematopoiesis, FLT3 signaling up-regulates MCL-1 but not BCL-2 and BCL-xL in HSCs and myeloid progenitors, promoting their cell survival.¹⁰ Therefore, we hypothesized that the constitutive expression of FLT3 signaling by FLT3-ITD mutation can induce up-regulation of MCL-1 in the AML LSC fraction. As shown in Figure 2B, the level of MCL-1 mRNA in FLT3-ITD AML BM cells was higher than that of FLT3-WT AML by approximately 2-fold, suggesting that the presence of FLT3-ITD can mark a fraction of AML with a high level of MCL-1. The high-level expression of MCL-1 protein in FLT3-ITD AML blasts was confirmed by Western blot analysis (Figure 2C-D). FLT3-ITD AML cells expressed approximately 7- and approximately 3-fold higher levels of MCL-1 protein compared with normal BM and FLT3-WT AML cells, respectively. In human hematopoiesis, HSCs express the highest level of MCL-1, and its expression gradually declines as they differentiate into mature myeloid or lymphoid cells.¹⁰ We therefore further subfractionated leukemic BM cells into CD34⁺CD38⁻ LSCs and CD34⁺CD38⁺ leukemic progenitor cell fractions. We could obtain sufficient numbers of cells for such analysis in 16 samples (10 cases of FLT3-WT and 6 cases of FLT3-ITD AMLs). As shown in Figure 3A, the CD34⁺CD38⁻ LSC fraction in FLT3-WT AMLs expressed approximately 6-fold higher, whereas those in FLT3-ITD AMLs had approximately 14-fold higher level of MCL-1, even compared with normal HSCs. In contrast, levels of BCL-2 and BCL-xL in the CD34⁺CD38⁻ LSCs and normal HSCs were not significantly different (Figure 3A). Cytoplasmic staining of MCL-1 FLT3-ITD AML LSC fraction shows that this population uniformly expressed MCL-1 protein at a high level on FACS, indicating that all single cells possessed high levels of MCL-1 (Figure 3B). Collectively, the CD34⁺CD38⁻ LSC fraction expressed the highest levels of MCL-1 in the leukemic BM.

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Figure 2

The expression of MCL-1 in FLT3-ITD and FLT3-WT AML cells. The levels of Mcl-1 transcripts and proteins in AML bone marrow cells. (A) AML cells express ~ 5-fold higher levels of MCL-1 compared with normal BM cells irrespective of their FAB types by real-time PCR assays. (B) FLT3-ITD AML cells expressed ~ 2-fold higher levels of MCL-1 in average compared with those in FLT3-WT AML cells. (C) The level of MCL-1 protein on Western blot analysis in primary AML cells. Results are shown as mean ± SD (*P < .05). (D) Representative Western blot data of MCL-1 in FLT3-WT AML (cases 1, 5, and 8) and FLT3-ITD AML (cases 23, 28, and 29) samples. Vertical lines have been inserted to indicate a repositioned gel lane.

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Figure 3

MCL-1 expression and its regulation in AML LSCs. (A) Real-time PCR analysis of MCL-1, BCL-2, and BCL-xl expression within fractions of CD34⁺ cells. The LSC-enriched CD34⁺CD38⁻ fraction in FLT3-WT AML (cases 1, 3-5, 8-10, 16, 18, and 19) expressed Mcl-1 at a level > 6-fold higher than that of normal HSCs, whereas LSCs in FLT3-ITD AML (cases 20, 22-24, 27, and 30) expressed even ~ 2-fold higher levels of MCL-1 compared with FLT3-WT LSCs. In contrast, BCL-2 or BCL-xL expression was not significantly changed. (B) Cytoplasmic staining of MCL-1 in CD34⁺CD38⁻ AML fractions with FLT3-ITD and FLT3-WT. A high amount of MCL-1 protein is detectable in FLT3-ITD LSCs. Representative FLT3-ITD and FLT3-WT LSCs are shown. (C) Changes in the expression level of MCL-1 in normal HSCs, LSCs of FLT3-WT AML, and of FLT3-ITD AML after incubation with FL or with SCF. Each bar represents an n-fold difference in the amount of MCL-1 expression prior to stimulation of normal HSCs. Results are mean ± SD (*P < .05).

FLT3 signaling stimulates MCL-1 expression in AML LSCs

Both FLT3 and c-KIT belong to the class III receptor tyrosine kinase family and share their major signaling cascade. Signals from either receptor play a critical role in maintenance of cell survival in normal HSCs and myeloid progenitors such as common myeloid progenitors and GMPs through MCL-1 up-regulation.¹⁰ In fact, both FLT3 and c-KIT were expressed in the CD34⁺CD38⁻ LSC fraction, and their expression levels appear to be equal in FLT3-ITD and FLT3-WT AMLs (Figure 1). We thus tested whether ligation of FLT3 and c-KIT receptors can induce further up-regulation of MCL-1 in AML LSCs in 5 cases each of FLT3-WT and FLT3-ITD AMLs. As shown in Figure 3C, both FLT3 ligand and SCF, a ligand for c-KIT, significantly up-regulated MCL-1 in LSCs. This effect was observed in both FLT3-WT and FLT3-ITD AMLs. In contrast, BCL-2 or BCL-xL expression was not significantly changed (not shown). A combination of FLT3 ligand and SCF did not show further additive effects on MCL-1 levels on LSCs (not shown). These data strongly suggest that signals of tyrosine kinase receptor family such as FLT3 and c-KIT can stimulate MCL-1 transcription in AML LSCs, presumably to prevent their apoptotic cell death.

MCL-1 is required for FLT3-ITD leukemic cells to maintain cell viability

Leukemic cell lines with FLT3-ITD, such as MV4-11, were used to analyze antiapoptotic functions of FLT3-ITD. The level of MCL-1 transcripts and proteins in MV4-11 cells was higher than that in normal BM controls, or other cell lines without FLT3 mutations such as NB4, HL-60, and U937 (Figure 4A), suggesting that FLT3-ITD signals up-regulate MCL-1 expression. MV4-11 was then treated with the tyrosine kinase inhibitor, PKC-412. PKC-412 down-regulated the expression of MCL-1 protein in a dose-dependent fashion (Figure 4B top). At the concentration of 0.5μM, MCL-1 was progressively inhibited (Figure 4B top), and reached plateau at 48 hours (not shown). PKC-412 induced apoptotic cell death of MV4-11 cells 24 hours after initiation of culture in a dose-dependent manner (Figure 4B bottom).

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Figure 4

Functional analysis of MCL-1 in FLT3-ITD AML. (A) MCL-1 expression in cell lines with FLT3-WT (NB4, HL-60, U937) and FLT3-ITD (MV4-11 and MOLM-13). High levels of MCL-1 mRNA and protein were seen in MV4-11 cells by real-time PCR analysis (left panel), the intracellular MCL-1 staining, and Western blot analysis (right panels). (B) The inhibition of MCL-1 protein (top) and cell survival (bottom) in MV4-11 after PKC-412 treatment. The inhibitory effect of PKC-412 reached plateau at a concentration of 0.5μM. Apoptotic cell death was evaluated by annexin/PI staining. Vertical lines have been inserted to indicate a repositioned gel lane. (C) The enforced expression of MCL-1 prevented FLT3-ITD cell lines from apoptosis in the presence of PKC-412. MCL-1 transcript and protein expression were significantly up-regulated 48 hours after transduction of MCL-1 into MV4-11 cells. PKC-412–induced apoptotic cell death is significantly inhibited in MV4-11 cells (right panel) (*P < .05). (D) Inhibition of MCL-1 by shRNA resulted in an apoptosis of FLT3-ITD cell lines. Forty-eight hours after transduction of MCL-1–shRNA–EGFP into the MV4-11 cells, MCL-1 transcript (left) as well as MCL-1 protein expression on FACS and Western blot analysis (middle) was significantly down-regulated. In parallel, the number of apoptotic cells increased by ~ 2-fold compared with control (right). Results are shown as mean ± SD (*P < .05). RE indicates relative expression levels.

We then tested whether reinforcement of MCL-1 can rescue FLT3-ITD cells from PKC-412–induced apoptosis. Expression vectors harboring MCL-1–enhanced green fluorescent protein and control EGFP were transduced into these FLT3-ITD AML cell lines. Forty-eight hours after transduction, an amount of MCL-1 mRNA in FLT3-ITD–EGFP⁺ cells was increased up to approximately 3-fold compared with those with a control vector (Figure 4C left). A significant increase in MCL-1 protein was also observed after transduction of MCL-1–EGFP vector (Figure 4C left). Purified FLT3-ITD–EGFP⁺ cells were incubated with graded concentrations of PKC-412 for 24 hours. PKC-412–induced apoptosis was significantly inhibited in cells transduced with MCL-1 transgene (Figure 4C right; supplemental Figure 1A, available on the Blood website; see the Supplemental Materials link at the top of the online article), suggesting that maintenance of MCL-1 level is critical for cell survival under this condition. We then inhibited MCL-1 expression in FLT3-ITD AML cell lines by transducing an shRNA expression vector. Vectors having MCL-1–shRNA–EGFP or control EGFP were transduced and the sorted live cells (EGFP⁺) were analyzed at 48 hours (Figure 4D). The amount of MCL-1 transcript reduced to approximately 60% of a control level. MCL-1 protein expression was also significantly reduced on FACS and Western blot analyses (Figure 4D middle). In parallel, numbers of apoptotic cells within MCL-1–shRNA–EGFP⁺ cells were increased up to approximately 2-fold compared with those in a control (Figure 4D right; supplemental Figure 1B). These results collectively suggest that FLT3-ITD supports leukemic cell survival by up-regulating MCL-1 production.

We appreciate the medical and nursing staff working on the Fukuoka Blood and Marrow Transplantation Group.

This work was supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology in Japan (19659248 and 20390272 [T.M.], 17109010 and 17047029 [K.A.]) and the Takeda Science Foundation (T.M.).