A pharmacodynamic study of the FLT3 inhibitor KW-2449 yields insight into the basis for clinical response

Abstract

Internal tandem duplication mutations of FLT3 (FLT3/ITD mutations) are common in acute myeloid leukemia (AML) and confer a poor prognosis. This would suggest that FLT3 is an ideal therapeutic target, but FLT3 targeted therapy has produced only modest benefits in clinical trials. Due to technical obstacles, the assessment of target inhibition in patients treated with FLT3 inhibitors has been limited and generally only qualitative. KW-2449 is a novel multitargeted kinase inhibitor that induces cytotoxicity in Molm14 cells (which harbor an FLT3/ITD mutation). The cytotoxic effect occurs primarily at concentrations sufficient to inhibit FLT3 autophosphorylation to less than 20% of its baseline. We report here correlative data from a phase 1 trial of KW-2449, a trial in which typical transient reductions in the peripheral blast counts were observed. Using quantitative measurement of FLT3 inhibition over time in these patients, we confirmed that FLT3 was inhibited, but only transiently to less than 20% of baseline. Our results suggest that the failure to fully inhibit FLT3 in sustained fashion may be an underlying reason for the minimal success of FLT3 inhibitors to date, and stress the importance of confirming in vivo target inhibition when taking a targeted agent into the clinical setting.

Trial registration: ClinicalTrials.gov NCT00346632.

Figures

Figure 1

KW-2249 and its metabolite inhibit FLT3. (A) Molm14 cells in culture medium were incubated for 1 hour in increasing concentrations of KW-2449 (top blots) and M1 (bottom blots). The cells were lysed, and FLT3 was immunoprecipitated from the extracts and resolved by SDS-PAGE. After probing the blots with antiphosphotyrosine (4G10), the blots were stripped and reprobed with anti-FLT3 to confirm equal lane loading. The blots were analyzed by densitometry, and the IC50 values were calculated using linear regression analysis. Vertical lines have been inserted to indicate a repositioned gel lane. (B) Extracts from the experiment in panel A were resolved directly with SDS-PAGE and the blots were probed with anti–phospho-STAT5, then stripped and reprobed with anti-STAT5 to confirm equal lane loading. Densitometry was performed as in panel A. Vertical lines have been inserted to indicate a repositioned gel lane. (C) Molm14 cells were incubated in culture medium with increasing concentrations of KW-2449 (solid line) or M1 (dashed line) for 48 hours. The MTT assay was then performed, with results plotted as percent untreated control. (D) Primary AML cells isolated from the peripheral blood of patients on the clinical trial prior to beginning treatment with KW-2449 were incubated for 48 hours with increasing concentrations of KW-2449. After 48 hours, the MTT assay was performed and the results plotted as percent untreated control. Four of the patients harbored FLT3/ITD mutations (solid lines), and 4 did not (dashed lines).

Figure 2

Inhibition of FLT3 in plasma. Molm14 cells were incubated for 1 hour in plasma containing increasing concentrations of KW-2449 (solid lines) and M1 (dashed lines). The cells were then lysed and analyzed for phosphorylated FLT3 (A) and STAT5 (C) as in Figure 1A and B. Densitometry was performed and the results plotted as percent untreated control in panels B and D. IC50 values were calculated using linear regression analysis.

Figure 3

The PIA assay is a valid surrogate of in vivo target inhibition for KW-2449. The immunoblot shows phospho-FLT3 (top) and total FLT3 (blot stripped and reprobed; bottom). A patient enrolled on the clinical trial of KW-2449 was administered a dose of 200 mg orally. Whole blood was obtained prior to the dose and at 2, 8, and 12 hours afterward, and separated into plasma and cellular fractions. Circulating blasts were isolated and analyzed for phospho-FLT3 as described in “Methods.” To perform the PIA assay, Molm14 cells were incubated for 1 hour in the plasma from the same time points, and likewise analyzed for phospho-FLT3 (as described in “Methods”). The first 4 lanes of the blot are from the circulating blasts, whereas the next 4 lanes show FLT3 from the Molm14 cells exposed to the plasma from which those blasts were isolated.

Figure 4

PIA results for patients receiving KW-2449. Plasma was isolated from whole-blood samples obtained from patients receiving increasing doses of KW-2449 on the clinical trial. Samples were obtained immediately prior to dosing, and at 2, 8, and 12 hours after dosing on days 1 and 14 of cycle 1. Dose levels 2, 4, and 6 correspond to total daily doses of 50, 200, and 400 mg, respectively (Table 1). Shown are the results from 3 representative patients on successively higher dose levels using the PIA assay for phosphorylated FLT3 (left) and STAT5 (right). Vertical lines have been inserted to indicate a repositioned gel lane.

Figure 5

PIA results compared with standard curve for KW-2449. (A) Plasma was collected from patients receiving KW-2449 on the clinical trial 2, 8, and 12 hours after ingesting drug. The plasma samples underwent conventional pharmacokinetic analysis for concentrations of KW-2449 and the metabolite, M1. In parallel, plasma from the same time points was used in PIA assays for FLT3. On the x axis, the concentration of active drug is plotted for individual time point by the following formula: [KW-2449] + [M1] / 3.6. On the y axis, the degree of FLT3 inhibition is plotted as percent control. The pretreatment plasma sample for each patient was used as the control for each individual PIA assay. Thus, each circle on the graph represents a single time point for which the concentration of KW-2449 plus metabolite in a plasma sample is plotted against how well that plasma inhibited FLT3 phosphorylation in Molm14 cells (the PIA assay). (B) This graph is the standard curve of KW-2449 inhibiting FLT3 in plasma shown in Figure 2. (C) This graph shows the graph from panel A overlying the standard curve from panel B, demonstrating how the results of the PIA assay, using the concentrations of KW-2449 and M1 divided by 3.6, correspond to the standard curve. (D) The same experiment as in panel C, but using the PIA assay for STAT5 rather than FLT3.

Figure 6

Plasma levels of KW-2449 and the metabolite M1. Plasma was obtained 2, 4, 8, and 12 hours after dosing on day 1 and day 14 of cycle 1 of the clinical trial for all patients enrolled. A “composite” drug level was determined by adding the concentration of KW-2449 with the concentration of the metabolite divided by 3.6 ([M1] / 3.6). The 3.6 divisor was derived from the standard curve experiments described in Figure 2. The graphs display the means values of these composite numbers at each of 7 dose levels (25, 50, 100, 200, 300, 400 and 500 mg per day), with error bars representing the standard error of the mean. The dashed line at 500 nM represents the level of drug estimated to be necessary in order to achieve a significant cytotoxic effect via FLT3 inhibition. (A) Day 1. (B) Day 14.

Figure 7

Intermittent inhibition of FLT3 in vivo. (A) Parallel flasks of Molm14 cells were incubated in culture medium containing escalating doses of KW-2449. For one set (4 hours, twice daily; dashed line), cells were exposed to KW-2449 for 4 hours, then washed in warm drug-free medium and resuspended in DMSO control medium. This was repeated twice daily for a total of 52 hours (5 total “doses”). Control cells (continuous; solid line) were treated in continuous fashion. The continuously treated cells were likewise washed twice daily, but then resuspended in KW-2449. One set of cells had drug incubated on the cells for 4-hour periods twice daily and the other set had continuous exposure to KW-2449. At the 52-hour time point each flask was analyzed by Trypan blue, MTT, and annexin V (data not shown for Trypan blue or annexin V). The results display reduced cytotoxicity with intermittent exposure to KW-2449. (B) Peripheral blood blasts were isolated by Ficoll gradient centrifugation (“Methods”) from a 60-year-old woman with relapsed/refractory AML harboring an FLT3/ITD mutation and incubated in culture medium containing increasing concentrations of KW-2449. After 48 hours, the MTT assay was performed. Blasts were isolated at the beginning of cycle 1 and at the beginning of cycle 2. (C) PIA results for cycle 1 for this patient, showing phosphorylated FLT3 and STAT5 on days 1 and 14 for different time points. Vertical lines have been inserted to indicate a repositioned gel lane. (D) Graph of WBCs over time. The horizontal black lines denote the time period during which the patient received treatment with KW-2449. The trial dictated a 5- to 7-day break from therapy between cycles. The patient was taken off study for progressive disease after completing the second cycle. (E) FLT3 phosphorylation in circulating blasts. These immunoblots show P-FLT3 immediately prior to, and 2 hours after, dosing with KW-2449 during the first and second cycles. Densitometry was performed and the density of the 2-hour time point was expressed as a percent of the pretreatment sample.