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. 2009 Oct 15;114(16):3459-63.
doi: 10.1182/blood-2007-10-113969. Epub 2009 Aug 25.

Acute dasatinib exposure commits Bcr-Abl-dependent cells to apoptosis

Jennifer L Snead  1 , Thomas O'Hare, Lauren T Adrian, Christopher A Eide, Thoralf Lange, Brian J Druker, Michael W Deininger
Affiliations

Acute dasatinib exposure commits Bcr-Abl-dependent cells to apoptosis

Jennifer L Snead et al. Blood. .

Abstract

Pioneering work with the Bcr-Abl inhibitor, imatinib, demonstrated a requirement for constant Bcr-Abl inhibition to achieve maximal therapeutic benefit in treating chronic myeloid leukemia (CML), establishing a paradigm that has guided further drug development for this disease. Surprisingly, the second-generation Bcr-Abl inhibitor, dasatinib, was reported to be clinically effective with once-daily dosing, despite a short (3- to 5-hour) plasma half-life. Consistent with this observation, dasatinib treatment of progenitor cells from chronic-phase CML patients for 4 hours, followed by washout, or continuously for 72 hours both resulted in an induction of apoptosis and a reduction in the number of clonogenic cells. Such acute treatments with clinically achievable dasatinib concentrations also irreversibly committed Bcr-Abl+ CML cell lines to apoptotic cell death. Potent transient Bcr-Abl inhibition using the alternative inhibitor, nilotinib, also resulted in cell death. These findings demonstrate that in vitro assays designed to model in vivo pharmacokinetics can predict clinical efficacy. Furthermore, they challenge the widely held notion that continuous target inhibition is required for optimal efficacy of kinase inhibitors.

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Figures

Figure 1
Figure 1
Response of Bcr-Abl–positive CML cells to treatment duration with various concentrations of dasatinib. LAMA-84 (A), KYO-1 (B), and K562 (C) cells were treated with 0, 10, 25, 50, or 100 nM dasatinib for the duration indicated, followed by washout, and monitored at 72 hours for annexin V staining. Averages ± SEM are reported for multiple independent experiments (LAMA-84, n = 3; KYO-1, n = 2; K562, n = 2). (D-F) Bcr-Abl activity was monitored with an antibody to phosphorylated CrkL (pY207) after treatment of LAMA-84 (D), KYO-1 (E), and K562 (F) cells with 0, 10, 25, 50, or 100 nM dasatinib for the duration indicated and for 4 hours after inhibitor washout. Total CrkL was examined in parallel as a total protein control. Experiments were performed at least twice for each cell line, and a representative dataset is shown.
Figure 2
Figure 2
Response of Bcr-Abl–positive CML cells to treatment duration with various concentrations of nilotinib. LAMA-84 (A), KYO-1 (B), and K562 (C) cells were treated with 0, 100, 500, 1000, or 5000 nM nilotinib for the duration indicated, followed by washout, and monitored at 72 hours for annexin V staining. Averages ± SEM are reported for multiple independent experiments (LAMA-84, n = 3; KYO-1, n = 2; K562, n = 2). (D-F) Bcr-Abl activity was monitored with an antibody to phosphorylated CrkL (pY207) after treatment of LAMA-84 (D), KYO-1 (E), and K562 (F) cells with 0, 100, 500, 1000, or 5000 nM nilotinib for the duration indicated and for 4 hours after inhibitor washout. Total CrkL was examined in parallel as a total protein control. Experiments were performed at least twice for each cell line, and a representative dataset is shown.
Figure 3
Figure 3
Response of primary CML progenitor cells to treatment duration with dasatinib. Progenitor cells isolated from 3 chronic-phase CML patients (CML 1-3) and a blast-crisis CML patient with 100% Bcr-AblT315I (CML R) were cultured in 100 nM dasatinib with (+) or without (−) washout at 4 hours, as indicated, or in the absence of dasatinib as an untreated control. K562 cells were treated identically to primary cells for comparison (gray bars, mean ± SEM from 2 independent experiments). (A) Percentage annexin V+ cells at 72 hours. Drug-specific apoptosis was calculated by subtracting the percentage annexin+ cells in the untreated arm. (B) Proliferation over 72 hours calculated as the fold expansion of viable cells during the 72-hour culture period, expressed as a percentage of untreated control. For K562, mean ± SEM of 2 independent experiments is reported. (C) Bcr-Abl activity at 24 hours, as measured by total phosphotyrosine level per cell and expressed as a percentage of the level in untreated cells. MFI indicates mean fluorescence intensity. (D) Proliferation of granulocyte/macrophage colony-forming cells. The number of cells at 72 hours that could lead to colony formation were enumerated and expressed as a percentage of untreated control. Colony-forming cell number was assayed in triplicate and mean ± SEM reported. (E) Bcr-Abl activity at 24 hours in CML 3 as assayed by CrkL phosphorylation. Western blot of total CrkL (bottom panel) showing phosphorylated (top band) and unphosphorylated (bottom band) forms. Top and bottom bands were quantified and the percentage phosphorylated plotted. (B-E) Dotted line represents the value for the untreated control.
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