Clinical specimens

BM aspirates were collected on days 0, 15, and 29. Slides were fixed in methanol and stained with Wright-Giemsa stain (Harleco/VGD). A 100-cell manual differential was performed using standard clinical protocols. Unstimulated cultures of BM were performed overnight and slides were prepared. G-banding was carried out according to standard clinical protocols. Twenty metaphases were analyzed for each case. No additional growth factors were used for conventional cytogenetic analysis. For analysis of cell morphology, cytospins were prepared using 1 × 10⁵ cells, which were centrifuged onto a glass slide and then fixed and stained with modified Wright-Giemsa stain (Sigma-Aldrich). Cells were analyzed by light microscopy using an Olympus BX41 clinical microscope. Photographs were taken using an attached Olympus DP72 digital camera with Olympus cellSens Version 1.3 software. For isolation of neutrophils, whole blood was centrifuged over a layer of Ficoll-Paque PLUS (GE Healthcare), and then the monolayer was extracted, overlaid onto Histopaque (Sigma-Aldrich), and centrifuged a second time. The monolayer was then isolated, washed, and cytospins were prepared. Light microscopy was used to confirm that the isolated cells were more than 95% neutrophils.

Cell culture and reagents

All cell lines and primary blast samples were cultured as described previously.¹⁹ Molm14 cells were obtained from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany). Sorafenib and quizartinib were dissolved in DMSO at stock concentrations of 10mM. Quizartinib was supplied by Ambit Biosciences. Sorafenib was obtained from LC Laboratories.

BM stroma coculture

Leftover BM from healthy donor harvests was collected, resuspended in RPMI medium (Invitrogen), and cells were centrifuged over a layer of Ficoll-Paque PLUS (GE Healthcare). Mononuclear cells were collected, washed twice with RPMI medium, and counted (Beckman Coulter). Cells were resuspended in stroma medium consisting of RPMI medium, 10% FBS, 1% L-glutamine, 1% penicillin-streptomycin (both Invitrogen), and 1μM hydrocortisone (Sigma-Aldrich). Cells were then plated into a 75-cm² tissue culture flask with an average number of 50 × 10⁶ cells/flask and were grown at 33°C in 5% CO₂. After 24-48 hours, medium containing nonadherent cells was removed and replaced with 20 mL of fresh stroma medium. Cells were cultured until 95%-100% confluence was reached. Cultures were passaged every 4-8 weeks after harvesting with 0.25% trypsin-EDTA (Sigma-Aldrich). For BM stroma coculture experiments, stromal cells were plated into 6-well plates (BD Biosciences) and allowed to grow until 95%-100% confluence was reached. Adherent cells were then washed with PBS 3 times and medium was replaced with culture medium. Molm14 cells or primary patient blasts were then added to the culture medium and plates were incubated at 37°C in 5% CO₂.

DNA isolation and sequencing

DNA was isolated from primary patient blasts using the DNeasy Mini Kit (QIAGEN). DNA sequencing, PCR for FLT3, and estimation of FLT3 mutant allelic burden were performed as described previously.¹⁹ Direct sequencing of the PCR products covering the entire C/EBPα coding sequence was with primers described previously.^20,21

Flow cytometry

Flow cytometric immunophenotyping was performed as described previously²² on primary patient blasts grown on stromal coculture in the presence or absence of quizartinib. For cell-cycle experiments, cells were seeded in 6-well plates containing a confluent stromal cell monolayer at a density of 2 × 10⁶ suspension cells/mL and incubated with or without quizartinib. After 24 hours (Molm14 cells) or 48 hours (primary patient blasts), cells were harvested and analyzed for propidium iodide staining, as described previously.²³

Immunoblotting

Electrophoresis and immunoblotting for FLT3 and ERK were performed as described previously.¹⁹ Abs to MMP-9 and to lactoferrin were obtained from Cell Signaling Technology. Anti–C/EBPα and anti–phospho-C/EBPα Abs were obtained from Santa Cruz Biotechnology and Cell Signaling Technology, respectively.

NBT reduction

For nitroblue tetrazolium (NBT) reduction assays, reagents were obtained from Sigma-Aldrich. Whole blood taken from quizartinib-treated patients and healthy controls was collected in heparinized tubes and incubated with NBT solution with or without stimulant solution (bacterial extract) per the manufacturer's instructions. Blood smears were prepared using the manual wedge method and were stained according to the manufacturer's instructions. Cells treated with quizartinib in vitro were also incubated as described above, cytospins were prepared as described under “Clinical specimens,” and staining was performed per the manufacturer's instructions.

Neutrophils are derived from the FLT3/ITD blasts

As patients accrued to this clinical protocol, it became apparent that quizartinib might be inducing the blasts to undergo differentiation. We therefore isolated and characterized the neutrophils produced during a surge. We prepared purified peripheral blood neutrophils from a patient at the peak of the surge (Figure 2A). We then prepared genomic DNA from the isolated neutrophils and from blasts collected from this same patient before beginning quizartinib therapy. PCR of the FLT3 juxtamembrane domain from both samples yielded fragments that migrated with an identical pattern on ethidium-stained agarose (Figure 2B). DNA sequencing of the mutant bands (not shown) confirmed that these were identical 75-bp ITD mutations, establishing that the neutrophils were derived from leukemic progenitors. We next analyzed these same purified neutrophils for expression of FLT3, lactoferrin, and MMP-9 (gelatinase) by immunoblotting. On normal mature neutrophils, FLT3 is not expressed³⁰ and lactoferrin and MMP-9 are found within the granules.³¹ Figure 2C shows the expression of constitutively activated FLT3 in the pretreatment blasts of this patient but not in the isolated neutrophils, whereas lactoferrin and MMP-9 were appropriately expressed in both the patient-derived and control neutrophils. The leukemia-derived neutrophils exhibited normal oxidative burst activity in an NBT assay (Figure 2D).

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Figure 2

Neutrophils are derived from the leukemic blasts. (A) Graph of peripheral blood absolute neutrophil count from patient 4 during treatment with quizartinib. Neutrophils from day 60 were isolated to > 95% homogeneity as described in “Methods,” which was confirmed by cytospin (inset). (B) Genomic DNA samples isolated from pretreatment blasts, day 60 neutrophils, and neutrophils from a healthy donor were analyzed by PCR for the FLT3/ITD mutation, as described in “Methods.” Shown is an ethidium bromide–stained agarose gel. WT indicates wild-type. (C) Whole-cell lysates were prepared from Molm14 cells, pretreatment blasts from patient 4, day 60 neutrophils from patient 4, and neutrophils from a healthy donor. Lysates were analyzed for total FLT3, phosphorylated FLT3, lactoferrin, and MMP9, as described in “Methods.” (D) NBT reduction assay of peripheral blood collected from a healthy donor (“Control”) compared with a quizartinib-treated patient during the neutrophil surge. Cells “with stimulation” were exposed to bacterial extract to induce respiratory burst activity.

These data indicate that in 13 of 14 FLT3/ITD AML patients, treatment with quizartinib resulted in terminal granulocytic differentiation of BM blasts. In one patient (Table 2 patient 14), however, there was no evidence of differentiation at any time during treatment. Although this patient experienced complete clearance of peripheral blasts, no neutrophil surge occurred (Table 1), and there was no clinically significant reduction in BM blasts on day 15 or 29. The patient had a normal karyotype, so the ongoing block in differentiation could not be explained by translocation of a transcription factor such as core binding factor. However, mutation analysis of the CEPBα coding sequence revealed a b-ZIP domain mutation (insertion of 4 base pairs, ACCG, at position 1449 of the reference sequence U34070, leading to a frameshift at R287), expected to result in loss of C/EBPα-mediated cell-cycle control.^6,32 CEPBα is a well-described transcription factor central to the induction of granulocyte development and is associated with transcriptional activation of genes such as lactoferrin.³³ Mutations in CEPBα occur in approximately 10%-15% of normal karyotype AML,³⁴ and are known to act in a dominant-negative manner to disrupt granulocytic differentiation.⁶ The presence of this CEPBα mutation offered a potential explanation for this patient's lack of differentiation in response to FLT3 inhibition. In a second patient (Table 2 patient 2), a trans-activation domain CEPBα mutation (deletion of 17 base pairs, CCGCCCCGGAGCCGCTG, at position 731 of the reference sequence U34070, leading to a frameshift at P46) was detected at disease progression 4 months after beginning quizartinib treatment.

Differentiation of Molm14 cells in vitro

FLT3 inhibition has been reported to induce granulocytic differentiation in FLT3/ITD AML cells.^17,18 These studies postulated that FLT3/ITD signaling leads to phosphorylation and potential inactivation of C/EBPα on residue ser21 via P-ERK1/2, resulting in a differentiation block that could be overcome with FLT3 inhibition. However, these observations were made using in vitro models with FLT3/ITD-expressing cell lines in suspension culture. The differentiation that we observed in our patients occurred within the BM. Therefore, we sought to develop an in vitro model of FLT3/ITD AML differentiation that more closely mimicked the conditions of the BM microenvironment, with direct interactions between the leukemia and BM stromal cells. Monolayers of stromal cells from healthy BM donors were cocultured with Molm14 cells, an FLT3/ITD-expressing AML line used previously to study differentiation in response to FLT3 inhibition.^18,35 This type of model has been used to study hematopoiesis and chemoresistance conferred by the BM microenvironment.^36,37

To determine whether differentiation in response to FLT3 inhibition represents a class effect of FLT3 TKIs, we cultured Molm14 cells on stromal layers with increasing doses of 2 different FLT3 TKIs, sorafenib and quizartinib.^19,38–40 In suspension culture, Molm14 cells undergo apoptosis on exposure to sustained, potent FLT3 inhibition.¹³ In contrast, whereas the cells cocultured on stroma arrested in G₁ in response to the inhibitors, the degree of apoptosis induced by either drug was diminished (Figure 3A-B). After 24 hours of treatment, morphologic differentiation of the cells was evident in the presence of either quizartinib (Figure 3C) or sorafenib (not shown) at an equipotent FLT3-inhibitory concentration,¹⁹ and respiratory burst activity by NBT assay was present (Figure 3D). This difference in response to FLT3 inhibition in suspension culture compared with stromal coculture parallels the responses observed clinically, in which peripheral blasts are rapidly cleared whereas BM blasts remain viable but undergo differentiation. It should be noted that although quizartinib inhibits both FLT3 and c-KIT, the IC₅₀ for inhibition of c-KIT is 35nM (data not shown), whereas the IC₅₀ for FLT3 is approximately 1nM.⁴⁰ Accordingly, the differentiation effects from this drug are most likely because of FLT3 inhibition and, although FLT3 autophosphorylation was fully inhibited under the conditions of this experiment (Figure 3E), P-ERK was only partially inhibited, in contrast to what is routinely observed with FLT3/ITD cell lines in suspension culture.^18,41 C/EBPα was readily detectable and phosphorylated despite FLT3 inhibition (Figure 3E). Because a previous study in murine 32D cells expressing a FLT3/ITD receptor indicated that C/EBPα levels increased in response to FLT3 inhibition,¹⁷ we examined C/EBPα levels over 24 hours of quizartinib exposure (Figure 3F). C/EBPα (and P-C/EBPα) was maximally expressed during the first 4-8 hours of stromal contact and then decreased as the cells differentiated.

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Figure 3

Treatment of Molm14 cells with FLT3 inhibitors. (A-B) Molm14 cells were exposed to quizartinib (10nM) or sorafenib (100nM) in suspension culture or coculture with BM stroma and analyzed for annexin V binding (A) and propidium iodide staining (B) by flow cytometry. (C-D) Molm14 cells were cocultured with stroma in the presence and absence of 10nM quizartinib for 24 hours, and then cells were collected and analyzed for morphology (C) and NBT reduction activity (D). (E) Molm14 cells were cocultured on stroma in the presence and absence of 10nM quizartinib. After 1 hour, cells were collected and lysates were analyzed by immunoblotting as described in “Immunoblotting.” (F) Molm14 cells were cocultured with stroma and quizartinib at the indicated concentrations. Cells were collected after 0, 4, 8, 12, and 24 hours of drug exposure and analyzed by immunoblotting for phosphorylated and total C/EBPα.

C/EBPα knockdown impedes FLT3 inhibitor-induced differentiation

Because of the central role of C/EBPα in myeloid differentiation and because the single patient in our series who failed to display differentiation harbored a C/EBPα mutation, we predicted that the loss of C/EBPα in FLT3/ITD cells would prevent FLT3 inhibitor–induced differentiation. Molm14 cells cocultured with stroma differentiated in a dose-responsive fashion to quizartinib (Figure 4A). We then used siRNA treatment of the Molm14 cells to knockdown C/EBPα levels (Figure 4B), which resulted in a significant decrease in differentiation induced by quizartinib (Figure 4C). This experiment demonstrates the importance of C/EBPα in mediating differentiation induced by FLT3 inhibition.

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Figure 4

Knockdown of C/EBPa blocks the differentiation induced by FLT3 inhibition. (A) Molm14 cells were cocultured with stroma in the presence of increasing concentrations of quizartinib for 24 hours, and then cells were collected and analyzed for morphology. For each condition, 100 viable cells were counted and categorized as undifferentiated or differentiated. (B) Molm14 cells (2 million per sample) were incubated with siRNA for C/EBP-α (or scrambled control), electroporated, and then cocultured with stroma. After 24 hours, the cells were harvested, lysed, and analyzed for C/EBPα protein levels by Western blotting. Incubation for 48 hours led to identical results (not shown). (C) Molm14 cells were subjected to siRNA C/EBPa knockdown as in panel B, cocultured with stroma for 24 hours, and then treated with 5nM quizartinib. After 24 hours, the cells were harvested and examined and scored for morphologic changes as in panel A.

This work was supported by grants from the National Cancer Institute (Specialized Programs of Research Excellence [SPORE] leukemia grants P50 CA100632-06 and R01 CA128864) and the American Society of Clinical Oncology (to M.L.). M.L. is a Clinical Scholar of the Leukemia & Lymphoma Society.