Fms-like tyrosine kinase 3 (FLT3) is implicated in the pathogenesis of acute myeloid leukemia (AML). FLT3-activating internal tandem duplication (ITD) mutations are found in approximately 30% of patients with AML and are associated with poor outcome in this patient population. Quizartinib (AC220) has previously been shown to be a potent and selective FLT3 inhibitor. In the current study, we expand on previous observations by showing that quizartinib potently inhibits the phosphorylation of FLT3 and downstream signaling molecules independent of FLT3 genotype, yet induces loss of viability only in cells expressing constitutively activated FLT3. We further show that transient exposure to quizartinib, whether in vitro or in vivo, leads to prolonged inhibition of FLT3 signaling, induction of apoptosis, and drastic reductions in tumor volume and pharmacodynamic endpoints. In vitro experiments suggest that these prolonged effects are mediated by slow binding kinetics that provide for durable inhibition of the kinase following drug removal/clearance. Together these data suggest quizartinib, with its unique combination of selectivity and potent/sustained inhibition of FLT3, may provide a safe and effective treatment against FLT3-driven leukemia. Mol Cancer Ther; 12(4); 438–47. ©2013 AACR.

Fms-like tyrosine kinase 3 (FLT3) is a member of the type III receptor tyrosine kinase (RTK) family and is required during early hematopoiesis (1). Deregulation of FLT3 is often found in the blast cells of leukemias including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL; refs. 2, 3). Approximately 30% of patients with AMLs harbor gain-of-function FLT3 internal tandem duplication (ITD) mutations that drive constitutive activation of downstream signaling cascades and are associated with poor disease outcome (4–7). Beyond activating mutations, overexpression of FLT3 and/or co-expression of the endogenous FLT3 ligand (FL) represent additional mechanisms by which FLT3 may be pathogenically activated (8). Activation of FLT3 kinase results in FLT3 autophosphorylation and induction of multiple downstream signaling cascades including the Ras/MAPK, PI3K/Akt, and STAT5 pathways (1, 9–13). In general, cells that have activated FLT3 are thought to be “addicted” to FLT3 signaling for their survival and growth, thereby rendering them exquisitely sensitive to FLT3-targeted therapy (14).

Multiple small-molecule FLT3 inhibitors, including lestaurtinib, sunitinib, sorafenib, tandutinib, and midostaurin, have been evaluated clinically (2, 15–22). Although these inhibitors were shown to be efficacious against FLT3-activated cell lines in vitro and in preclinical efficacy models (19, 23–28), clinical results to date have been disappointing due to dose-limiting toxicity and lack of durable responses in patients with AMLs (22, 29). With the exception of tandutinib, these inhibitors are multi-kinase inhibitors that lack FLT3, or even RTK, selectivity (30). The undesirable off-target effects of these compounds may have prevented administration of sufficiently high doses to achieve complete and sustained FLT3 inhibition. Nevertheless, the few responses observed in patients with AMLs with these inhibitors were often accompanied by inhibition of constitutive FLT3 phosphorylation (15, 25, 31, 32), suggesting that more potent, selective, and durable inhibition of FLT3 may result in a broader and more significant response in patients with AMLs.

Quizartinib/AC220, is a small-molecule kinase inhibitor that potently inhibits FLT3 with a high degree of kinome selectivity (30). We have previously shown the potent inhibition by quizartinib of cellular FLT3 autophosphorylation and cell viability in the FLT3-ITD cell line, MV4-11, and the translation of this potent inhibition into effective antitumor activity in tumor xenograft models (30). A recent phase I clinical study of quizartinib in patients with AMLs indicates that continuous, once-daily administration of this drug can lead to reduced FLT3 signaling and tumor burden, as more than 50% of FLT3-ITD patients and a smaller percentage of patients without the ITD mutation, had a partial or complete response to treatment (unpublished data).

To better understand the mechanism of FLT3-mediated efficacy of quizartinib, we evaluated its inhibitory activity across different FLT3 isoforms, its impact on downstream FLT3 signaling pathways and the durability of this inhibition in vitro and in vivo. These studies reveal quizartinib to be a potent inhibitor of FLT3 signaling across a variety of FLT3 genotypes yet triggers apoptosis only in cells with endogenously activated FLT3. Furthermore, we show that slow quizartinib dissociation from FLT3 may cause sustained inhibition of FLT3 autophosphorylation and downstream signaling, induction of cell death in vitro, and achievement of a durable response in human tumor xenografts following a single administration. These results suggest that quizartinib may be unique among clinical FLT3 inhibitors with respect to potency, specificity, and durability of FLT3 inhibition.

Reagents

Quizartinib (FLT3; Kd = 1.6 nmol/L), lestaurtinib (FLT3; Kd = 8.5 nmol/L), midostaurin (FLT3; Kd = 11 nmol/L), and sorafenib (FLT3; Kd = 13 nmol/L) were synthesized at Ambit Biosciences (30). Tandutinib (FLT3; Kd = 3 nmol/L), was custom-synthesized by CiVentiChem, and sunitinib (FLT3; Kd = 0.5 nmol/L) was custom-synthesized by Sai Advantium, Ltd. (all Kd values from ref. 30). Antibodies for immunoblot analysis were purchased as follows: FLT3, extracellular signal–regulated kinase (Erk1/2), phospho-Erk1/2 (T202/Y204), and β-actin from Santa Cruz Biotechnology, Inc.; Akt, phospho-Akt (S473), PARP, cleaved PARP (D214), and phospho-STAT5 (Y694) from Cell Signaling Technology; STAT5 from BD Biosciences; and phosphotyrosine (pTyr; 4G10) from Millipore. Culture media were obtained from MediaTech and FBS from Omega Scientific. Except where noted, all other reagents were obtained from Sigma-Aldrich.

Cell culture

MV4-11, RS4;11, THP-1, and HEL 92.1.7 (HEL) cells were obtained from American Type Culture Collection (ATCC). Cell lines were authenticated at ATCC before purchase by their standard short tandem repeat DNA typing methodology. MOLM-14 and SEM-K2 cells were a gift from Dr. Mark Levis (Johns Hopkins Medical School, Baltimore, MD). Within 1 month of receipt, cultures were grown out for several passages and aliquots of each were frozen. For experimental use, each cell type was thawed and grown out for no more than 2 months before use in an experiment. The cells were maintained in 10% FBS in IMDM (MV4-11), RPMI-1640 (MOLM-14 and SEM-K2), and RPMI-1640 supplemented with d-glucose, HEPES, 2 mmol/L l-glutamine, sodium bicarbonate, and sodium pyruvate (HEL, RS4;11, and THP-1). Cells were serum-starved overnight in medium containing 0.5% FBS before treatment with compound for immunoblots and viability assays.

Immunoprecipitation of FLT3

Serum-starved cells were washed with cold PBS. RS4;11 cells were either untreated or treated for 15 minutes with 100 ng/mL human FL (R&D Systems) before the PBS wash. For quizartinib treatment, serum-starved MV4-11 cells were incubated with a dose titration of quizartinib for 2 hours. Twenty million MV4-11 cells were used for each dose. Cells were washed with cold PBS and lysed for 20 minutes on ice with Cell Extraction Buffer (Invitrogen) supplemented with protease (Roche Applied Science) and phosphatase (EMD Chemicals) inhibitor cocktails and 1 mmol/L phenylmethylsufonyl fluoride (PMSF). Following a 15-minute centrifugation to clear the lysate, 2 mg of lysate was incubated with FLT3 antibody for 16 hours at 4°C. Protein A-agarose was added to this mixture and incubated for 2 hours at 4°C. Bead complexes were washed 3 times with Cell Extraction Buffer and proteins eluted with NuPAGE LDS Sample Buffer (Invitrogen). FLT3 immunoprecipitates were analyzed by SDS-PAGE and immunoblotting for phospho-tyrosine and total-FLT3 content.

Immunoblot analysis

Immunoprecipitates or 50 μg of whole-cell lysates were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with primary antibodies overnight at 4°C. Fluorescent secondary antibodies (LI-COR Biosciences) used for detection were incubated for 30 minutes at room temperature. Blots were scanned using the LI-COR Odyssey Infrared Imaging System, and bands were quantified using this system's application software.

FLT3 and STAT5 immunoassays

Following overnight culture in low serum (0.5% FBS), cells were seeded at 400,000 cells per well in a 96-well plate and treated with inhibitors as described above. Following compound incubation, FLT3 phosphorylation was induced in RS4;11 and THP-1 cells with 100 ng/mL FL for 15 or 5 minutes, respectively. Cell lysates (Cell Lysis Buffer from Cell Signaling Technology supplemented with protease and phosphatase inhibitor cocktails and 1 mmol/L PMSF) were cleared by centrifugation. Phosphorylated and total STAT5a/b levels in the lysates were determined using the Multi-Spot Phospho-(Tyr 694); Total STAT5a/b assay from Meso Scale Discovery (MSD) as instructed by the manufacturer. Levels of phosphorylated and total FLT3 in the lysates were similarly determined using the MSD electrochemiluminescence platform as previously described (30). All IC50 calculations were conducted using IGOR Pro 5 software (WaveMetrics) on data normalized to dimethyl sulfoxide (DMSO)-treated cells.

Cell viability assays

Serum-deprived cells were seeded at a density of 50,000 (MV4-11), 30,000 (MOLM-14), or 60,000 (SEM-K2, RS4;11, and THP-1) cells per well in a 96-well plate and then treated with DMSO or a dose titration of inhibitors. CellTiter-Blue (CTB) reagent (Promega) was added to the cells after 3 or 7 days following inhibitor addition. The resulting fluorescent signals were measured after 3 hours using a fluorescence plate reader. Viability was calculated as a percentage of cells treated with DMSO.

PARP cleavage assays

Cells starved overnight were incubated with a dose titration of quizartinib for 24 hours (MV4-11), 6 days (SEM-K2), or 7 days (RS4;11). Cells were lysed and subjected to immunoblot analysis as described above for cleaved and uncleaved PARP.

Durability experiments with washouts

For CTB viability assays, cells treated as described above were incubated with drug for 30 minutes, 24 hours, or 48 hours, washed 3 times with PBS, followed by addition of fresh low-serum media and incubated such that the total assay time was 72 hours for each. Control cells were treated with compounds continuously for 72 hours with no washout. Viability was measured using CTB as described above. For phosphorylated FLT3 and STAT5 analyses, serum-starved cells were treated with compound for 2 hours washed with PBS as above, replenished with fresh media containing 0.5% FBS, then further incubated for 30 minutes, 6 or 24 hours before cell lysis and MSD immunoassay analysis as described above. Control cells with no recovery time were lysed immediately following the 2-hour compound treatment. Structures for the inhibitors used in these experiments (quizartinib, lestaurtinib, and sorafenib) are found in Fig. 5.

Kinetic binding assays

Binding assays were conducted as described previously (33). The assays measure the ability of a test compound to compete with the interaction between a kinase and a known ATP site—binding ligand that has been immobilized on a solid support using quantitative PCR. Binding reactions were incubated for 90 minutes and either left undiluted or diluted as indicated. These reactions were measured at 20 and 120 minutes post-dilution.

In vivo efficacy model

Female C.B-17 SCID mice (Harlan Laboratories) were implanted subcutaneously on day 0 with 5 × 106 MV4-11 cells in a 50:25:25 homogeneous mixture of PBS and High/Low concentration Basement Membrane Matrigel (BD Biosciences). Treatments began when the mean estimated tumor mass for all groups (10 mice per group) reached about 250 mm3. Quizartinib, formulated in 5% hydroxypropyl-β-cyclodextrin, was administered at 1 or 10 mg/kg by oral gavage once a day for 1 day or 14 consecutive days. Animals with tumors in excess of 2,000 mm3 or with excessive ulcerated tumors were euthanized as were those found in obvious distress or in a moribund condition. Body weights and tumor measurements were recorded 3 times weekly. Tumor burden was estimated from caliper measurements by the formula for the volume of a prolate ellipsoid assuming unit density as: tumor burden (mm3) = (L × W2)/2, where L and W are the respective orthogonal tumor length and width measurements in mm. Tumor growth inhibition (T/C, %) and tumor growth delay (T-C, days) were each calculated relative to the predefined endpoint of 1,500 mm3 as previously defined (34).

Tumor lysates

When tumor volume reached approximately 300 mm3, animals received a single dose of 1 or 10 mg/kg quizartinib by oral gavage. Tumors were harvested at various time points post-dose and were extracted with ice-cold Cell Extraction Buffer supplemented with protease and phosphatase inhibitor cocktails and 1 mmol/L PMSF using mechanical dissociation. Tumor extracts were analyzed for phosphorylated FLT3 by MSD immunoassay and for phosphorylated STAT5, AKT, ERK, and cleaved PARP levels by immunoblotting as described above.

Quizartinib is a potent inhibitor of FLT3 autophosphorylation in wild-type and ITD-mutated FLT3 cells

To explore the impact of FLT3 genotype on quizartinib-mediated FLT3 inhibition, we expanded on an earlier study (30) to include a panel of 5 leukemia cell lines expressing wild-type (WT) or ITD-mutated FLT3. MV4-11 cells are homozygous for the FLT3-ITD mutation, whereas MOLM-14 cells harbor both a WT and ITD allele (35). RS4;11, THP-1, and SEM-K2 cells are each homozygous for WT FLT3; however, SEM-K2 cells have an amplification of the FLT3 gene leading to elevated expression and constitutive activation of FLT3 (Fig. 1A and B). MV4-11 and MOLM-14 cells also have constitutively activated FLT3 as a result of the ITD mutation, whereas RS4;11 and THP-1 cells require FL for activation/phosphorylation of the receptor (Fig. 1B and data not shown). Similar to its effects on MV4-11 cells (30), quizartinib potently downregulated the autophosphorylation of FLT3 in MOLM-14 and SEM-K2 cells and FL-stimulated FLT3 phosphorylation in RS4;11 and THP-1 cells (Table 1 and Supplementary Fig. S1). While slightly higher IC50 values were observed in the FLT3-WT cells compared with the FLT3-ITD cells (4- to 9-fold), these results indicate that quizartinib is a potent inhibitor of FLT3 phosphorylation irrespective of genotype and ranks among the most potent when compared with a panel of FLT3 inhibitors (Supplementary Table S1).

Figure 1.

FLT3 expression profile in cell panel and quizartinib-mediated inhibition of STAT5, Erk, and Akt. A, expression levels of FLT3 receptor were tested by immunoblot analysis from untreated MV4-11, MOLM-14, and SEM-K2 and from RS4;11 cells treated for 15 minutes without or with 100 ng/mL FL (+FL). HEL cells were used for a negative control cell lysate. B, phospho-FLT3 in leukemia cell lines. FLT3 was immunoprecipitated from cells treated as in A and probed with anti-phosphotyrosine (pTyr; top) and anti-FLT3 (bottom) antibodies. C–F, MV4-11, MOLM-14, SEM-K2, and RS4;11 cells were treated with DMSO vehicle or the indicated amounts of quizartinib for 2 hours and the lysates were analyzed by immunoblot for total and phosphorylated STAT5, Erk1/2, and Akt. RS4;11 cells were stimulated with 100 ng/mL FL (+FL) for 15 minutes before cell lysis as indicated.

Figure 1.

FLT3 expression profile in cell panel and quizartinib-mediated inhibition of STAT5, Erk, and Akt. A, expression levels of FLT3 receptor were tested by immunoblot analysis from untreated MV4-11, MOLM-14, and SEM-K2 and from RS4;11 cells treated for 15 minutes without or with 100 ng/mL FL (+FL). HEL cells were used for a negative control cell lysate. B, phospho-FLT3 in leukemia cell lines. FLT3 was immunoprecipitated from cells treated as in A and probed with anti-phosphotyrosine (pTyr; top) and anti-FLT3 (bottom) antibodies. C–F, MV4-11, MOLM-14, SEM-K2, and RS4;11 cells were treated with DMSO vehicle or the indicated amounts of quizartinib for 2 hours and the lysates were analyzed by immunoblot for total and phosphorylated STAT5, Erk1/2, and Akt. RS4;11 cells were stimulated with 100 ng/mL FL (+FL) for 15 minutes before cell lysis as indicated.

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Table 1.

Signaling and cell viability profile for quizartinib

IC50, nmol/L
AssayMV4-11MOLM-14SEM-K2RS4;11THP-1
pFLT3 0.9 1.7 4.4 8.3 
pSTAT5 0.7 0.2 0.3 ND ND 
pErk 0.5 0.4 0.7 0.3 ND 
pAkt 0.6 0.3 0.7 3.1 ND 
Viability 0.3 0.1 0.4 >10,000 >10,000 
IC50, nmol/L
AssayMV4-11MOLM-14SEM-K2RS4;11THP-1
pFLT3 0.9 1.7 4.4 8.3 
pSTAT5 0.7 0.2 0.3 ND ND 
pErk 0.5 0.4 0.7 0.3 ND 
pAkt 0.6 0.3 0.7 3.1 ND 
Viability 0.3 0.1 0.4 >10,000 >10,000 

Abbreviation: ND, not determined.

Inhibition of FLT3-mediated signaling pathways

Constitutive and ligand-induced activation of FLT3 leads to phosphorylation of downstream effector molecules in signaling networks such as the STAT5, Ras/MAPK, and PI3K/Akt pathways. Consistent with FLT3 inhibition, quizartinib potently inhibited phosphorylation of constitutively activated STAT5, Erk1/2, and Akt in MV4-11, MOLM-14, and SEM-K2 cells with IC50 values in the range of 0.3 to 0.7 nmol/L, and of FL-stimulated phosphorylation of Erk1/2 and Akt in RS4;11 cells with IC50 values of 0.3 and 3 nmol/L, respectively (Fig. 1C–F, Table 1 and Supplementary Fig. S1). Consistent with previous reports (12, 13, 36), STAT5 is not constitutively phosphorylated, nor does FL induce its phosphorylation in RS4;11 cells (Fig. 1F).Taken together, these results show that quizartinib-mediated FLT3 inhibition, in either FLT3-ITD or–WT cells, is accompanied by potent attenuation of downstream signaling events.

Quizartinib inhibits cell survival in FLT3-activated cells

To determine whether FLT3 genotype would influence the sensitivity of cells to FLT3 inhibition with respect to cell viability, we assessed cell viability across the cell panel following treatment with quizartinib. Both of the FLT3-ITD cell lines, MV4-11 and MOLM-14, were exquisitely sensitive to quizartinib treatment with cell viability IC50 values of 0.1 to 0.3 nmol/L measured at 72 hours (Fig. 2A, Table 1). Interestingly, while an IC50 of 0.4 nmol/L could be measured after 72 hours in SEM-K2 cells, complete loss of viability required 5 to 7 days (Fig. 2A and B, Table 1). In contrast, minimal or no loss of viability was observed in the FLT3-WT–expressing RS4;11 and THP-1 cells (Fig. 2A and B, Table 1), even following 7 days of quizartinib treatment, consistent with reports that these lines are not dependent on FLT3 signaling for sustained cell growth (18, 37). Importantly, comparison of quizartinib to a panel of FLT3 inhibitors revealed similar rank order across the cell panel discussed above, with quizartinib showing the greatest FLT3 potency and selectivity toward FLT3-activated cells especially compared with some of the less FLT3-selective inhibitors (Supplementary Table S1).

Figure 2.

Differential sensitivity of leukemia cell viability and apoptosis upon quizartinib treatment. Cell viability was measured 3 (A) or 7 (B) days after compound addition. Percentage of viability was calculated on the basis of DMSO-treated control cells. C, immunoblot analysis of PARP cleavage in cells treated with DMSO or the indicated amounts of quizartinib for 24 hours (MV4-11 and MOLM-14), 6 days (SEM-K2), or 7 days (RS4;11). The percentage of cleaved PARP was calculated as the amount of cleaved PARP divided by the sum of cleaved PARP and full-length PARP.

Figure 2.

Differential sensitivity of leukemia cell viability and apoptosis upon quizartinib treatment. Cell viability was measured 3 (A) or 7 (B) days after compound addition. Percentage of viability was calculated on the basis of DMSO-treated control cells. C, immunoblot analysis of PARP cleavage in cells treated with DMSO or the indicated amounts of quizartinib for 24 hours (MV4-11 and MOLM-14), 6 days (SEM-K2), or 7 days (RS4;11). The percentage of cleaved PARP was calculated as the amount of cleaved PARP divided by the sum of cleaved PARP and full-length PARP.

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Quizartinib induces apoptosis in FLT3-activated cells

Treatment with quizartinib induced significant and dose-dependent PARP cleavage and accumulation of sub-2N DNA in MV4-11, MOLM-14, and SEM-K2 cells (Fig. 2C and Supplementary Fig. S2). Cleavage was observed as early as 8 (data not shown) and 24 hours after quizartinib treatment. Consistent with the effects of quizartinib on cell viability, significant PARP cleavage nor appearance of sub-2N DNA were observed in the SEM-K2 cells until 6 days posttreatment, whereas no increase in PARP cleavage or sub-2N DNA was observed in RS4;11 cells after 7 days of treatment (Fig. 2C and Supplementary Fig. S2).

Durability of quizartinib-mediated inhibition of FLT3 signaling

To assess the relative durability of quizartinib-mediated signaling inhibition in cell culture, we treated MV4-11 cells with quizartinib for 2 hours and then measured the levels of phosphorylated FLT3 (pFLT3) and STAT5 (pSTAT5) at 0.5, 6, and 24 hours after compound withdrawal (Table 2 and Supplementary Fig. S3). The approximately 1 nmol/L pFLT3 IC50 of quizartinib was maintained following a 0.5-, 6-, and even 24-hour washout period (Table 2). This durable inhibition translated to STAT5 phosphorylation as well, where no dramatic changes in IC50 values were observed under all 3 washout conditions tested (Table 2 and Supplementary Fig. S3). In contrast, lestaurtinib was found to be highly sensitive to the washouts resulting in about 3-, 4-, and 8-fold increase in pFLT3 IC50 following 0.5, 6, and 24 hours of drug removal (Table 2 and Supplementary Fig. S3). This translated to a 4-fold increase in the pSTAT5 IC50 values at the 6- and 24-hour washout. In parallel experiments, sorafenib showed a modest 2- to 3-fold increase in IC50 values for both pFLT3 and pSTAT5 (Table 2, Supplementary Fig. S3, and compound structures in Fig. 5).

Table 2.

IC50 values for washout experiments in MV4-11

Washout durationNo wash0.5 h6 h24 h
Compound incubation time
 pFLT3, IC50, nmol/L 
Quizartinib 1.1 0.9 1.3 1.1 
Lestaurtinib 2.0 5.6 8.2 15.9 
Sorafen 1.0 1.0 2.3 2.7 
pSTAT5, IC50, nmol/L 
Quizartinib 0.3 0.4 0.7 0.7 
Lestaurtinib 0.3 0.4 1.3 1.3 
Sorafenib 0.5 0.4 1.1 1.6 
 72 h 48 h 24 h 0.5 h 
Compound exposure time
 Cell viability, IC50, nmol/L 
Quizartinib 0.4 0.4 0.4 0.9 
Lestaurtinib 1.9 5.9 12.5 60.8 
Sorafenib 0.7 1.5 0.9 4.6 
Washout durationNo wash0.5 h6 h24 h
Compound incubation time
 pFLT3, IC50, nmol/L 
Quizartinib 1.1 0.9 1.3 1.1 
Lestaurtinib 2.0 5.6 8.2 15.9 
Sorafen 1.0 1.0 2.3 2.7 
pSTAT5, IC50, nmol/L 
Quizartinib 0.3 0.4 0.7 0.7 
Lestaurtinib 0.3 0.4 1.3 1.3 
Sorafenib 0.5 0.4 1.1 1.6 
 72 h 48 h 24 h 0.5 h 
Compound exposure time
 Cell viability, IC50, nmol/L 
Quizartinib 0.4 0.4 0.4 0.9 
Lestaurtinib 1.9 5.9 12.5 60.8 
Sorafenib 0.7 1.5 0.9 4.6 

Duration of compound exposure and loss of cell viability

To assess the impact of brief exposures on cell viability, MV4-11 cells were treated with compounds for various times, washed to remove inhibitors, and then assayed for viability 72 hours after initial exposure. Quizartinib exposures as short as 30 minutes led to virtually the same dose–response relationship as that achieved with continual 72 hours exposure (Table 2 and Supplementary Fig. S3). Under these conditions of brief quizartinib exposure, dose- and time-dependent increases in the pro-apoptotic BimEL were observed, along with decreases in the anti-apoptotic protein Mcl-1 (Supplementary Fig. S4). No changes in BimL BimS, Bax, Bcl-XL, or Bcl-2 were observed (Supplementary Fig. S4 and data not shown). Lestaurtinib was considerably more sensitive to compound removal with shorter exposures of lestaurtinib resulting in 3-, 7-, and 32-fold increases in IC50 for the 48-, 24-, and 0.5-hour exposures, respectively (Table 2 and Supplementary Fig. S3). Sorafenib-treated cells showed an intermediate sensitivity to compound removal (Table 2 and Supplementary Fig. 3). Similar durability properties were observed for each of these compounds in the MOLM-14 and SEM-K2 cells (data not shown). These results suggest that in cells dependent on constitutively activated FLT3, durable inhibition of FLT3 signaling following a brief exposure to quizartinib translates to lasting effects on cell viability mediated by modulation of pro- and anti-apoptotic proteins.

Time dependence of FLT3 binding

To understand how quizartinib mediates sustained FLT3 inhibition, we tested the effect of incubation time and dilution of the FLT3/inhibitor mixture on the apparent affinity of quizartinib to FLT3 using the kinase-binding technology described previously (33). To compare relative off-rates compounds were incubated with FLT3 for 1.5 hours followed by a series of dilutions and assayed for binding at 20 and 120 minutes. The binding curves generated from the diluted FLT3/quizartinib reactions were nearly identical to those of the undiluted reactions (Fig. 3A and B), as reflected in the 0.3 to 0.9 nmol/L Kd for all dilutions, and are indicative of a slow dissociation rate for quizartinib. In contrast, dilution of the FLT3/lestaurtinib binding reaction resulted in a clear shift in the binding curves corresponding to >30- and >100-fold increases in apparent Kd between no dilution and 50-fold dilution of the lestaurtinib/FLT3 mixture at each time point and is consistent with a rapid off-rate for lestaurtinib (Fig. 3C and D). Taken together, these binding data suggest that a slower quizartinib off-rate compared with lestaurtinib likely contributes to the differential effects these compounds show relative to FLT3 inhibition and cell viability described above.

Figure 3.

Time-dependent binding of quizartinib to FLT3. Quizartinib (A and B) and lestaurtinib (C and D) were preincubated with FLT3 for 1.5 hours and then diluted as indicated. The binding was measured either 20 minutes (A and C) or 2 hours (B and D) after dilution.

Figure 3.

Time-dependent binding of quizartinib to FLT3. Quizartinib (A and B) and lestaurtinib (C and D) were preincubated with FLT3 for 1.5 hours and then diluted as indicated. The binding was measured either 20 minutes (A and C) or 2 hours (B and D) after dilution.

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In vivo efficacy and durability of FLT3 inhibition

To determine whether the durability properties of quizartinib in vitro translated to in vivo efficacy, we tested single-dose administrations of either 1 or 10 mg/kg quizartinib compared with the same doses given once per day for 14 days in the previously described MV4-11 xenograft model (30). Daily administration of 10 or 1 mg/kg quizartinib for 14 days led to significant tumor reduction consistent with our previous observations (Fig. 4A and Supplementary Table S2; ref. 30). Following the dosing period, tumor growth in the 1 mg/kg cohort rebounded rapidly (TGD = 21 days), whereas the 10 mg/kg cohort showed sustained tumor remission up to 30 days following the final dose of quizartinib (TGD = 55 days). Remarkably, after a single 10 mg/kg dose of quizartinib, no tumor growth was observed for up to 2 weeks (TGD = 15 days). Even a single 1 mg/kg dose of quizartinib caused significant reduction in tumor growth (TGD = 5 days; Fig. 4A and Supplementary Table S2).

Figure 4.

Durability of efficacy with a single dose of quizartinib in MV4-11 xenograft tumors. A, mice bearing MV4-11 xenograft tumors were treated with drug vehicle or 1 or 10 mg/kg of quizartinib orally once daily (QD) for 1 or 14 days. B, time course of FLT3, STAT5, Erk1/2, or Akt phosphorylation levels in MV4-11 tumor lysates from (n = 3) animals treated with a single oral dose of 1 or 10 mg/kg of quizartinib. C, immunoblot analysis of PARP cleavage in tumor treated with vehicle or a single dose of 1 or 10 mg/kg quizartinib.

Figure 4.

Durability of efficacy with a single dose of quizartinib in MV4-11 xenograft tumors. A, mice bearing MV4-11 xenograft tumors were treated with drug vehicle or 1 or 10 mg/kg of quizartinib orally once daily (QD) for 1 or 14 days. B, time course of FLT3, STAT5, Erk1/2, or Akt phosphorylation levels in MV4-11 tumor lysates from (n = 3) animals treated with a single oral dose of 1 or 10 mg/kg of quizartinib. C, immunoblot analysis of PARP cleavage in tumor treated with vehicle or a single dose of 1 or 10 mg/kg quizartinib.

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Figure 5.

Chemical structures of FLT3 inhibitors.

Figure 5.

Chemical structures of FLT3 inhibitors.

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To determine whether differences in tumor volume reduction between single doses of 1 versus 10 mg/kg quizartinib correlated with pharmacodynamic assessments, levels of pFLT3, pErk1/2, pSTAT5, and pAkt at various times following drug administration were assessed in tumor lysates. Quizartinib administration leads to a rapid decrease in FLT3, STAT5, and Erk1/2 phosphorylation in the tumors at both doses tested (Fig. 4B). The 1 mg/kg dose reduced FLT3 phosphorylation by 60% at 2 hours, reached maximal inhibition of approximately 70% by 6 hours and had recovered to within 20% of control levels by 24 hours. STAT5 phosphorylation followed a similar pattern following the 1 mg/kg dose with 40% inhibition at 2 hours, 75% inhibition at 6 hours, and full recovery at 24 hours. Inhibition of Erk1/2 phosphorylation was not as robust with inhibition levels of 30% at both 2 and 6 hours and control levels returning by 24 hours. Akt phosphorylation was not impacted following a 1 mg/kg dose of quizartinib. At 10 mg/kg, the impact on signaling was much more pronounced with 80% inhibition of pFLT3 observed at both the 2- and 6-hour time points and a residual 50% inhibition at 24 hours. Similarly, pErk1/2 levels were reduced by roughly 70% at 2 and 6 hours, returning to within 30% inhibition by 24 hours. More striking was the effect of 10 mg/kg on STAT5 phosphorylation, which was reduced by more than 95% at all 3 time points. Consistent with the effect at the 1 mg/kg dose, the 10 mg/kg dose only modestly inhibited (10%–15%) Akt phosphorylation at 2 and 6 hours with pAkt levels elevated at 24 hours.

Finally, to determine whether the rapid loss of tumor volume was associated with an induction of apoptosis, cleavage of PARP was followed in tumor lysates. Tumors from mice receiving either 1 or 10 mg/kg showed considerable PARP cleavage 2 and 6 hours posttreatment with the peak of cleavage occurring at the 6-hour time point (Fig. 4C). In the 1 mg/kg tumor lysates, no additional PARP cleavage was observed at 24 hours, whereas in the 10 mg/kg lysates, PARP cleavage remained elevated. These experiments suggest that the degree of sustained antitumor activity achieved appears to correlate with the durability of FLT3 signal inhibition within the first 24 hours and may reflect durable inhibition of FLT3 in residual tumor cells.

Although clinical activity has been observed with first-generation FLT3 inhibitors, as a class they have failed to fulfill the promise of providing long-term therapeutic benefit in AMLs (15–17, 21, 22, 29). Potential explanations for these marginal clinical results include insufficient FLT3 potency leading to residual disease and/or inadequate FLT3 selectivity leading to off-target effects and associated dose-limiting toxicity (2, 14, 22). Among responders to these first-generation FLT3 inhibitors, patients with FLT3-ITD mutations were in the clear majority; however, a smaller number of ITD-negative patients also responded to therapy (38, 39). In the preclinical studies presented here, we characterize quizartinib with respect to potency and durability of inhibition against WT and ITD-mutant FLT3 kinase across a panel of cells harboring distinct FLT3 genotypes, compare these properties to a panel of first- and second-generation FLT3 inhibitors, and show that quizartinib possesses slow off-rate kinetics. These studies showed that (i) quizartinib potently inhibits cellular signaling in both ITD and WT cells, yet translation to induction of apoptosis was restricted to cells with constitutive FLT3 activation, whether ITD or WT; (ii) as a result of slow off-rate kinetics, quizartinib requires only transient exposures to effect durable inhibition of FLT3, its downstream signaling pathways, and ultimately cell viability in FLT3-dependent cells in vitro and in vivo; and finally, (iii) quizartinib is unique among current FLT3 inhibitors in this profile of selectivity across the kinome, potency against cellular FLT3, and durability of inhibition once achieved.

The ability of quizartinib to distinguish FLT3-dependent from FLT3-independent cells may be important for 2 reasons. First, this profile suggests that quizartinib therapy may spare cells not directly reliant upon FLT3 activity for survival. As not all FLT3 inhibitors are able to make this distinction (Supplementary Table S1 and ref. 30), tolerability issues associated with these inhibitors may be attributed to off-target–mediated induction of apoptosis in non–FLT3-dependent cells. Second, quizartinib-induced apoptosis in FLT3-WT–activated cells (SEM-K2) is reminiscent of the clinical experience where a significant minority of responding patients lacked the FLT3 ITD. Defining the molecular mechanisms responsible for this FLT3 dependency in the ITD-negative population is the subject of current preclinical and clinical investigation.

A common challenge in the clinical development of kinase inhibitors has been the inability to maintain prolonged target inhibition without exceeding tolerated levels of the drug. While TKIs including lestaurtinib and midostaurin have shown transient clinical responses, other inhibitors such as sorafenib and dasatinib have shown more durable responses with target inhibition sustained after efficacious levels of drug have been eliminated in vivo or washed away in vitro (17, 22, 40–42). In the studies reported here, reduction of FLT3 phosphorylation and downstream signaling is maintained for up to 24 hours after quizartinib is removed from culture, and an exposure time as low as 30 minutes was as effective as a 72-hour incubation to induce cell death in sensitive cells. In vivo, this durability contributes to sustained inhibition of FLT3, STAT5, and ERK phosphorylation after a single dose, and long after the majority of the administered quizartinib has been eliminated from peripheral circulation. Our binding studies suggest that slow dissociation of quizartinib from FLT3 may contribute to enhanced efficacy relative to a simple pharmacokinetic “time-over-target” estimation. Similar to our findings with quizartinib, slow inhibitor off-rates have been shown to mediate durable target inhibition for other kinase inhibitors (43).

Quizartinib appears to be unique among the panel of tested inhibitors on several accounts. Although lestaurtinib and sorafenib showed similar potency to quizartinib with respect to FLT3 inhibition, lestaurtinib and to a lesser degree sorafenib retained significant activity against the non–FLT3-activated cells (ref. 30 and Supplementary Table S1). This theme was repeated with respect to durability of inhibition in that quizartinib showed significantly enhanced durability of efficacy and slower binding kinetics relative to lestaurtinib. In a recent report comparing quizartinib with lestaurtinib in blasts derived from a small cohort of patients with AMLs, the authors suggest that a less selective FLT3 inhibitor such as lestaurtinib may be more effective at first diagnosis than a more selective inhibitor such as quizartinib (44). While confirmation of this hypothesis awaits a side-by-side comparison of clinical results, it is quite plausible that the degree to which blasts are addicted to FLT3 signaling (and therefore respond to a FLT3-selective inhibitor) may vary in the heterogeneous AML patient population, especially at first diagnosis. Regardless of the potential for a more modest initial response compared with the multi-kinase inhibitors, the prolonged efficacy after a brief exposure in vivo, along with its potency, durability, and selectivity in vitro, may bode well for the safe, durable, and efficacious use of quizartinib in the clinic.

No potential conflicts of interest were disclosed.

Conception and design: R.N. Gunawardane, B. Belli, R.C. Armstrong

Development of methodology: R.N. Gunawardane, R.R. Nepomuceno, J.P. Hunt, B. Belli, R.C. Armstrong

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R.N. Gunawardane, R.R. Nepomuceno, A.M. Rooks, J.P. Hunt, J.M. Ricono

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R.N. Gunawardane, R.R. Nepomuceno, J.P. Hunt, J.M. Ricono, B. Belli, R.C. Armstrong

Writing, review, and/or revision of the manuscript: R.N. Gunawardane, R.R. Nepomuceno, J.P. Hunt, J.M. Ricono, R.C. Armstrong

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R.N. Gunawardane, R.C. Armstrong

Study supervision: R.N. Gunawardane, B. Belli, R.C. Armstrong

The authors thank M. Holladay, P. Zarrinkar, and W. Wierenga for thoughtful discussions and critical reading of the manuscript and R Faraoni, M. Hocker, and B. Campbell for synthesis of midostaurin, lestaurtinib, sorafenib, and quizartinib.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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