Motesanib inhibits Kit mutations associated with gastrointestinal stromal tumors

Unless specified otherwise all reagents were purchased from Sigma Aldrich; all cell culture reagents were purchased from Invitrogen (Carlsbad, CA).

Female C57B6 mice (6 to 8 weeks old; 20 to 30 g; Charles River Laboratories, Wilmington, MA) were anesthetized, and an area of skin 2 × 2 cm on the right flank was depilated. Oral administration of either 75 mg/kg motesanib (Amgen Inc., Thousand Oaks, CA) or vehicle (water, pH 2.5) was initiated on the same day as depilation and continued for 21 days. On day 21, photographs were taken for assessment of hair depigmentation. The same patch of skin was depilated again on day 28, and photographs for assessment of depigmentation were taken on day 35. All animal experimental procedures were conducted in accordance with the guidelines of the Amgen Animal Care and Use Committee and the Association for Assessment and Accreditation of Laboratory Animal Care standards.

KIT mutants (Table 1) were identified from published reports [8] and generated using PCR-based site-directed mutagenesis. PCR products were cloned into the pcDNA3.1+ hygro vector or the pDSRα22 vector (Amgen Inc), gel purified, and then ligated with a common 5' fragment of human wild-type KIT to yield full-length, mutant constructs in pcDNA3.1+ hygro or pDSRα22 expression vectors.

Fluorescence activated cell sorting (FACS) was utilized to isolate pools of CHO and Ba/F3 cells stably expressing wild-type and mutant KIT variants. FACS was performed on a FACS Aria cell sorter (BD Biosciences San Jose, CA), under sterile conditions using 488 nm laser excitation. KIT transfected cells were labeled with the anti-Kit monoclonal antibody SR1 (prepared at Amgen Inc.; data on file) followed by incubation with FITC-labeled secondary anti-mouse IgG antibody (SouthernBiotech, Birmingham, AL). Cells were then resuspended in Dulbecco's phosphate-buffered saline with 0.5% bovine serum albumin at a final concentration of 1 × 10⁶ cells per mL to ensure a constant and viable sorting rate of 5000 cells/sec. Cells transfected with vector control were used to adjust the baseline instrument settings. Forward and side scatter gating enabled the exclusion of dead cells and debris. The top 10% to 15% of Kit-positive cells within the overall transfected cell population were then isolated to ensure collection of high-expressing cells. Cells were sorted directly into 15 mL conical tubes containing the appropriate growth media. Cell pools were then cultured and maintained under the respective selection conditions, and were reanalyzed for Kit expression prior to characterization of Kit autophosphorylation.

CHO cells stably transfected with wild-type or mutant isoforms of KIT were seeded in a 96-well tissue culture plate at a density of 2 × 10⁴ cells per well. For stem cell factor (SCF) characterization experiments, cells were stimulated with serial dilutions of SCF for varying times. To determine IC₅₀ values, the cells were treated for 2 hours with single 10-fold serial dilutions of motesanib or imatinib starting at 3 μM. Cell lines transfected with wild-type KIT were stimulated for 10 minutes with 100 ng/mL SCF following treatment with motesanib or imatinib. Cell lines transfected with activating KIT mutants were not stimulated with SCF in IC₅₀ experiments. Cells were washed with phosphate-buffered saline and lysed in RIPA buffer (50 mM Tris, pH 7; 150 mM NaCl, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS, 300 μM activated sodium vanadate, 1× protease inhibitor cocktail) for 30 minutes at 4°C with shaking. Cell lysates were added to a 96-well DELFIA microplate (PerkinElmer Inc.) coated with anti-Kit antibody (1 μg per well; AF332, R&D Systems, Inc.; Minneapolis, MN) and incubated for 2 hours. Lysates were then removed and the plate was washed 3 times with DELFIA wash buffer (PerkinElmer Inc.). Recombinant anti-phosphotyrosine antibody 4G10 (Cat. # 05-777; Upstate/Millipore, Billerica, MA) was added to each well (0.1 μg per well) and incubated at room temperature for 1 hour. The plate was then washed 3 times with DELFIA wash buffer before 0.01 μg of Eu-N1-labeled anti-mouse antibody (Cat. # AD0124, PerkinElmer Inc.) was added to each well. The plate was again incubated at room temperature for 1 hour and then washed 3 times with DELFIA wash buffer before the signal was detected by adding DELFIA enhancement buffer (PerkinElmer Inc.) to each well. Luminescence was measured using a Victor Model 1420 multilabel counter (PerkinElmer Inc.). Kit autophosphorylation at each motesanib or imatinib concentration was expressed as a percentage of the vehicle control (0.2% DMSO).

The ability of Kit mutants to act as survival factors was assessed in Kit-dependent Ba/F3 cells. Ba/F3 cells stably transfected with various KIT mutants were seeded in a 96-well tissue culture plate at a density of 5 × 10³ cells per well. To determine IC₅₀ values, cells were treated for 24 hours with single 10-fold serial dilutions of motesanib or imatinib starting at 3 μM (0.1 μM for motesanib-treated V560 D and Δ552-559 Kit mutants). Cell viability was assessed by measuring the level of adenosine triphosphate using ATPlite assays (PerkinElmer Life Sciences, Boston, MA). Reconstituted ATPLite 1-step solution was added to each well followed by incubation with shaking for 2 minutes. The plate was read on a Victor Model 1420 multilabel counter (PerkinElmer Inc.) under the luminescence setting. Viability at each motesanib or imatinib concentration was expressed as a percentage of the vehicle control (0.2% DMSO).

Motesanib potently inhibited SCF-induced autophosphorylation of Kit in CHO cells stably transfected with the wild-type KIT gene (IC₅₀ = 36 nM). In comparison, imatinib inhibited wild-type Kit with an IC₅₀ of 165 nM.

Hair depigmentation was used as a surrogate marker to assess the ability of motesanib to inhibit Kit activity in vivo [16]. Following depilation, female C57B6 mice were administered either 75 mg/kg motesanib (n = 8) or vehicle (n = 8) twice daily for 21 days. In mice receiving motesanib, hair regrowth was markedly depigmented compared with mice receiving vehicle (Figure 1). This effect was reversible. Following the cessation of motesanib treatment on day 21, the mice were depilated again on day 28. There was no apparent depigmentation of regrown hair on day 35. Similar results were obtained in male mice (data not shown).

Figure 2 summarizes the results from the autophosphorylation experiments using CHO cells stably transfected with the wild-type KIT gene or various KIT mutant genes. Tyrosine phosphorylation of wild-type Kit was dose-dependent, with the greatest intensity of autophosphorylation occurring after a 30 minute incubation of the cells with 300 ng/mL of SCF. In contrast, tyrosine phosphorylation of activated Kit mutants occurred in the absence of SCF with no further phosphorylation induced by treatment with SCF.

In CHO cells, motesanib inhibited the autophosphorylation of the primary activating Kit mutants V560 D, Δ552-559, and AYins503-504 (Table 2; Figure 3B). In each instance, motesanib was a more potent inhibitor of Kit autophosphorylation than imatinib. For example, motesanib inhibited the AYins503-504 mutant with an IC₅₀ of 18 nM, whereas imatinib inhibited this mutant with an IC₅₀ of 84 nM. Interestingly, the IC₅₀ values for inhibition of these Kit mutants were lower than the IC₅₀ for inhibition of wild-type Kit by motesanib. Consistent results were obtained in a functional viability assay utilizing IL-3-independent growth of Ba/F3 cells (Figure 3C). For example, when testing the AYins503-504 mutant, the IC₅₀ for motesanib was 11 nM versus 47 nM for imatinib.

Motesanib inhibited the activity of Kit mutants associated with secondary imatinib resistance. In Kit autophosphorylation assays, motesanib inhibited tyrosine phosphorylation of the juxtamembrane domain/kinase domain I double mutants V560D/V654A and V560D/T670I with IC₅₀ values of 77 nM and 277 nM, respectively. Imatinib had limited activity against the V560D/V654A mutant and no activity against the V560D/T670I mutant at concentrations of up to 3000 nM (Table 3; Figure 4B). Consistent results were obtained in the Ba/F3 cells expressing the V560D/V654A and V560D/T670I mutants with motesanib IC₅₀ values of 91 nM and 180 nM, respectively. Again, motesanib was a more potent inhibitor of these mutants than imatinib (Table 3; Figure 4C).

Similarly, motesanib inhibited autophosphorylation of the imatinib-resistant activation loop mutant Y823 D (IC₅₀ = 64 nM) more potently than imatinib (IC₅₀ > 3000 nM) (Table 3: Figure 4B). However, neither motesanib nor imatinib inhibited autophosphorylation of the D816V mutant (Table 3). Consistent with these results, motesanib inhibited the growth of Ba/F3 cells transfected with the V560D/V654A, V560D/T670I, or Y823 D mutant more potently than imatinib. Of note, the IC₅₀ of imatinib against the Y823 D mutant when established in the functional viability assay was at least 10-fold lower than the IC₅₀ measured in the autophosphorylation assay. IL-3-independent Ba/F3 cells expressing the D816V Kit mutant could not be established.