Activating mutations of the activation loop of KIT are associated with certain human neoplasms, including the majority of patients with systemic mast cell disorders, as well as cases of seminoma, acute myelogenous leukemia (AML), and gastrointestinal stromal tumors (GISTs). The small-molecule tyrosine kinase inhibitor imatinib mesylate is a potent inhibitor of wild-type (WT) KIT and certain mutant KIT isoforms and has become the standard of care for treating patients with metastatic GIST. However, KIT activation loop mutations involving codon D816 that are typically found in AML, systemic mastocytosis, and seminoma are insensitive to imatinib mesylate (IC50 > 5-10 μmol/L), and acquired KIT activation loop mutations can be associated with imatinib mesylate resistance in GIST. Dasatinib (formerly BMS-354825) is a small-molecule, ATP-competitive inhibitor of SRC and ABL tyrosine kinases with potency in the low nanomolar range. Some small-molecule SRC/ABL inhibitors also have potency against WT KIT kinase. Therefore, we hypothesized that dasatinib might inhibit the kinase activity of both WT and mutant KIT isoforms. We report herein that dasatinib potently inhibits WT KIT and juxtamembrane domain mutant KIT autophosphorylation and KIT-dependent activation of downstream pathways important for cell viability and cell survival, such as Ras/mitogen-activated protein kinase, phosphoinositide 3-kinase/Akt, and Janus-activated kinase/signal transducers and activators of transcription. Furthermore, dasatinib is a potent inhibitor of imatinib-resistant KIT activation loop mutants and induces apoptosis in mast cell and leukemic cell lines expressing these mutations (potency against KIT D816Y ≫ D816F > D816V). Our studies suggest that dasatinib may have clinical efficacy against human neoplasms that are associated with gain-of-function KIT mutations. (Cancer Res 2006; 66(1): 473–81)
Gain-of-function mutations of the KIT receptor tyrosine kinase play an important role in oncogenesis of certain human malignancies, including the vast majority of gastrointestinal stromal tumors (GISTs; refs. 1, 2) and a subset of hematologic neoplasms ( 3– 7) and germ cell tumors ( 8, 9). KIT is a class III receptor tyrosine kinase and is structurally characterized by an extracellular domain with five immunoglobulin-like repeats, a single transmembrane domain, a juxtamembrane domain, and a cytoplasmic tyrosine kinase domain. The kinase domain consists of the NH2-terminal (TK1) and COOH-terminal (TK2) lobes that are separated by a hydrophilic kinase insert. The TK2 domain contains the kinase activation loop, a critical hinged region of the kinase that must assume a particular conformation to allow full kinase activation ( 10).
Imatinib mesylate is a potent KIT tyrosine kinase inhibitor ( 11) and is now the standard frontline therapy for advanced GISTs ( 2, 12). Although imatinib is a potent inhibitor of the kinase activity of wild-type (WT) KIT and GIST-associated juxtamembrane domain mutant KIT isoforms, most KIT activation loop mutations are resistant to clinically achievable doses of imatinib ( 2, 11– 16). Imatinib only binds to the inactive conformation of KIT; however, KIT activation loop mutations not only activate kinase activity but also stabilize the activation loop in a conformation that does not allow productive imatinib binding ( 10, 17, 18). Activating KIT activation loop mutations are found in association with acute myelogenous leukemia (AML; ref. 5), mast cell disease (in particular systemic mastocytosis; refs. 4, 13), a subset of sinonasal natural killer/T-cell non-Hodgkin lymphoma ( 6, 7), seminoma/dysgerminoma ( 8, 9), and imatinib-resistant GIST ( 2, 12).
Dasatinib, formerly known as BMS-354825, is an ATP-competitive, dual SRC/ABL inhibitor ( 19). Notably, dasatinib can inhibit BCR-ABL activation loop mutations that are found in some chronic myelogenous leukemia (CML) patients with acquired clinical resistance to imatinib ( 20). Some small-molecule SRC/ABL inhibitors also have potency against KIT kinase ( 11, 14, 21, 22). Therefore, we hypothesized that dasatinib might inhibit the kinase activity of both WT and mutant KIT isoforms.
We report herein that dasatinib potently inhibits WT KIT and juxtamembrane domain mutant KIT autophosphorylation and KIT-dependent activation of downstream pathways important for cell viability and cell survival, such as Ras/mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase/Akt, and Janus-activated kinase/signal transducers and activators of transcription (STAT). Furthermore, we show that dasatinib is a potent inhibitor of imatinib-resistant KIT activation loop mutants and induces apoptosis in mast cell and leukemic cell lines expressing these mutations.
Materials and Methods
Cell lines. The WT FLT3 Ba/F3 cell line, a murine interleukin 3 (IL-3)–dependent hematopoietic pro-B cell line, the Chinese hamster ovary cell line Chinese hamster ovary-K1, and the murine p815 mast cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA). The murine KIT D814Y mutant isoform expressed by the p815 cell line ( 23) is homologous to the human KIT D816Y mutation. The human hematopoietic growth factor–dependent M-07e cell line was obtained from Dr. Hal Broxmeyer (Department of Microbiology and Immunology, Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN). The human HMC-1.1 mast cell line expressing a KIT juxtamembrane domain mutant isoform (V560G) was kindly provided by Dr. Butterfield (Division of Allergic Diseases, Department of Internal Medicine, Mayo Clinic, Rochester, MN). A spontaneously occurring subclone of the HMC-1.1 cell line, HMC-1.2 ( 24, 25), which has an additional mutation in the activation loop (D816V), was kindly provided by Dr. Akin (Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD). All cell lines were grown in RPMI 1640 supplemented with 10% fetal bovine serum (HyClone, South Logan, UT), 1% penicillin G (10,000 units/mL), and streptomycin (10,000 μg/mg), 2 mmol/L l-glutamine (both Life Technologies-Invitrogen, Carlsbad, CA). Filtered IL-3 containing supernatant (10%) from WEHI-3 cells (ATCC) was added to the growth medium for the parental Ba/F3 cell line. M-07e cells were cultured using recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) as a growth supplement as previously described ( 11).
Site-directed mutagenesis and generation of a Ba/F3 cell line expressing mutant KIT. KIT cDNA was generously provided by Dr. Axel Ullrich (Department of Molecular Biology, Max Planck Institute for Biochemistry, Martinsried, Munich, Germany) and cloned into the pLXSN retroviral vector plasmid (BD Biosciences, Palo Alto, CA), the pCDNA3.1 vector plasmid, or the M5gNeo plasmid ( 26). Site-directed mutagenesis was used to create the D816V, D816Y, D816F mutations (QuickChange kit, Stratagene, La Jolla, CA) and all mutations were confirmed by bidirectional sequencing ( 27). Retroviral transduction was done and Ba/F3 cell lines stably expressing mutant KIT isoforms were generated by double selection for G418 resistance and IL-3-independent growth ( 28– 30).
Transient transfections of CHO-K1 Chinese hamster cell lines with KIT WT or mutant isoforms were done using a lipofection assay (LipofectAMINE kit purchased from Life Technologies-Invitrogen). Cells were treated with dasatinib 24 hours after transfection ( 2).
Antibodies and reagents. An anti-KIT rabbit polyclonal antibody, an anti-STAT3 mouse monoclonal antibody (both Santa Cruz Biotechnology, Santa Cruz, CA), an anti-AKT (polyclonal) rabbit antibody (Cell Signaling Technology, Beverly MA), and an anti-MAPK1/2 [extracellular signal-regulated kinase 1/2 (ERK1/2)] rabbit monoclonal antibody (Upstate Biotechnology, Lake Placid, NY) were used at a 1:5,000 to 1:1,000 dilution. Antiphosphotyrosine KIT antibodies (Tyr568/570 and Tyr703), an antiphosphothreonine/tyrosine MAPK (Thr202/Tyr204) antibody, an antiphosphothreonine (Thr308) and an antiphosphoserine (Ser473) AKT antibody, an antiphosphotyrosine (Tyr705) STAT3 antibody, and a pan-antiphosphotyrosine antibody (clone PY20) were used at dilutions of 1:100 to 1:2,000 (all from Cell Signaling Technology). Peroxidase-conjugated goat anti-mouse antibody and goat anti-rabbit antibody were used at 1:5,000 and 1:10,000 dilutions, respectively (Bio-Rad, Hercules, CA). Protein A/G PLUS-Agarose immunoprecipitation reagent was purchased from Santa Cruz Biotechnology. The small-molecule compound dasatinib (formerly BMS-354825) was obtained from Bristol-Myers Squibb (Princeton, NJ). Imatinib mesylate (STI571/Gleevec) was purchased from the Oregon Health Science University Hospital pharmacy (Portland, OR). Imatinib and dasatinib were dissolved in DMSO to create 10 mmol/L stock solutions and stored at −20°C.
Western blots. Cells (∼5 × 107) were exposed to varying concentrations of dasatinib and cultured for 90 minutes at 37°C in a 5% CO2 atmosphere. Cell pellets were lysed with 100 to 150 μL of protein lysis buffer (50 mmol/L Tris, 150 mmol/L NaCl, 1% NP40, 0.25% deoxycholate with added inhibitors aprotinin, AEBSF, leupeptin, pepstatin, sodium orthovanadate, and sodium pyruvate). Protein from cell lysates (500-2,000 μg) was used for immunoprecipitation experiments and 75 to 200 μg protein from cell lysates were used for whole cell protein analysis by Western immunoblot assays as previously described ( 28).
Proliferation assays. Cells were added to 96-well plates at densities 30,000 cells per well. Dasatinib was added and proliferation was measured at 72 hours using an 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT)–based assay (Roche Molecular Biochemicals, Indianapolis, IN; ref. 11).
Apoptosis assays. Cells were incubated with dasatinib for 48 to 72 hours and translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane as an early indicator of apoptosis was analyzed using an Annexin V-FITC kit (Immunotech, Marseilles, France) and a FACScalibur flow cytometer loaded with CellQuest analysis software (BD, Heidelberg, Germany; ref. 11).
Data analysis. Dose-effect plots were created to calculate the IC50 for the treatment effect of dasatinib for each cell line (Calcusyn Software available from Biosoft, Cambridge, United Kingdom; ref. 29).
Dasatinib inhibits the kinase activity of WT and juxtamembrane domain mutant KIT isoforms. Dasatinib is a potent dual SRC/ABL kinase inhibitor that is currently in phase I/II clinical studies of CML and solid tumors. Based on structural homology considerations and prior descriptions of the activity of some SRC and/or ABL inhibitors against KIT ( 11, 14, 21, 22), we hypothesized that dasatinib might also inhibit KIT kinase activity. Indeed, dasatinib potently inhibited the ligand-dependent autophosphorylation of WT KIT kinase in the cytokine-dependent human myeloid leukemia cell line M-07e with an IC50 of 1 to 10 nmol/L. Dasatinib also inhibited stem cell factor (SCF)–dependent proliferation of these cells with a similar IC50 (5-10 nmol/L). In comparison, the IC50 values for imatinib inhibition of autophosphorylation and proliferation were 50 to 100 nmol/L, respectively ( Fig. 1A-B ; ref. 31). Dasatinib had little effect on the GM-CSF-dependent proliferation of these cells (IC50 > 10,000 nmol/L), suggesting that the effect of dasatinib on SCF-dependent proliferation was due to its inhibition of KIT kinase rather than direct effects on downstream kinases (e.g., SRC family members) that might be common to both the KIT and GM-CSF receptors ( Fig. 1B).
Gain-of-function mutations involving the KIT juxtamembrane domain occur in some cases of mast cell disease ( 24) and AML ( 5). In addition, KIT juxtamembrane domain mutations are found in approximately two thirds of GISTs, and this GIST subset has the best clinical response to imatinib ( 11, 12, 32). We tested the activity of dasatinib against KIT juxtamembrane domain mutations using the HMC-1.1 cell line, which is a spontaneously immortalized human mast cell leukemia cell line that expresses the KIT V560G mutant isoform ( 24). This particular mutation is one of the most common juxtamembrane domain point mutations found in GISTs ( 2). Dasatinib inhibited the kinase activity of KIT V560G in a dose-dependent manner with an IC50 of ∼10 nmol/L, which is nearly identical to the previously reported results for imatinib ( Fig. 2A ; refs. 11, 16). Imatinib potently inhibited cellular proliferation and induced apoptosis of this cell line in the low nanomolar range ( Fig. 2; ref. 11). Therefore, we tested whether dasatinib had similar biological effects. Both dasatinib and imatinib inhibited cellular proliferation of HMC-1.1 cells with an IC50 of 5 to 10 nmol/L ( Fig. 2B). As shown in Fig. 2C, dasatinib induced apoptosis of HMC-1.1 cells with an IC50 of 14 nmol/L, whereas the IC50 for imatinib was ∼70 nmol/L. These data indicate that in HMC-1.1 cells, KIT kinase activation is required for cellular proliferation and survival and dasatinib is at least as potent as imatinib for inhibiting the KIT V560G juxtamembrane domain mutation.
Dasatinib inhibits the kinase activity of imatinib-resistant KIT activation loop mutations found in hematologic malignancies. Although imatinib potently inhibits the kinase activity of WT and juxtamembrane domain mutant KIT isoforms, it has minimal activity against KIT D816Y, D816F, or D816V mutant kinases ( 13). The inability of imatinib to inhibit these mutant isoforms is due to steric clash between imatinib and the “open” (or active) conformation of the KIT activation loop ( 19, 20). The predicted structural model of dasatinib binding to ABL suggests that changes in activation loop conformation might not significantly affect drug binding ( 19). We hypothesized that dasatinib would also be less sensitive to the KIT activation loop conformational changes than imatinib and, therefore, dasatinib would more potently inhibit KIT activation loop mutations involving codon 816. To test this hypothesis, we used a spontaneously generated subclone of the HMC-1.1 cell line HMC-1.2, which acquired the typical mastocytosis-associated D816V mutation on the same allele as the original V560G mutation ( 24, 25). KIT is constitutively autophosphorylated in this cell line but is resistant to treatment with clinically relevant doses of imatinib ( 13). Consistent with previous studies, the IC50 of imatinib for cellular proliferation of this cell line was >10,000 nmol/L ( Fig. 3A-B ; refs. 13, 24).
In contrast, dasatinib inhibited the kinase activity of KIT V560G/D816V in a dose-dependent manner with an IC50 of 50 to 100 nmol/L. Despite inhibition of KIT kinase activity by dasatinib in the low nanomolar range, the compound was nearly 1 log less potent for inhibition of cellular proliferation and induction of apoptosis, with IC50 values of 1,200 and 2,000 nmol/L, respectively ( Fig. 3A-C). These results suggest that HMC-1.2 cells are less dependent on KIT kinase activity for cellular proliferation and survival than the parental HMC-1.1 cells, possibly due to other oncogenic events that occurred during the emergence of this subclone.
We tested the activity of dasatinib against the spontaneously occurring murine mastocytosis cell line, p815, which expresses a murine KIT D814Y mutation that is homologous to the human D816Y mutation. Dasatinib potently inhibited KIT autophosphorylation with an IC50 of 1 to 10 nmol/L and inhibited the cellular proliferation and induced apoptosis of p815 cells with IC50 values of 10 to 25 and ∼25 nmol/L, respectively ( Fig. 3D-F). Therefore, unlike our results with the HMC-1.2 cells, dasatinib inhibition of KIT kinase in p815 cells was strongly correlated with inhibition of cellular proliferation and induction of apoptosis. Notably, high-dose imatinib (>1,000 nmol/L) inhibited KIT autophosphorylation. A dose of 1,200 nmol/L imatinib inhibited the proliferation of p815 cells by 30% but did not significantly induce programmed cell death.
Effects of different amino acid substitutions of KIT aspartic acid 816 (D816) on sensitivity to dasatinib. Our results with the HMC-1.2 and p815 cell lines suggested that dasatinib might have different potency against D816Y than against D816V mutations. Alternatively, these results could reflect differences in activity of dasatinib against human or murine KIT and/or differences in drug uptake by the different cell lines. To address this issue, we generated isogenic factor–independent Ba/F3 cell lines expressing systemic mastocytosis–associated codon 816 mutations with an interchange of aspartic acid to valine (D816V), tyrosine (D816Y), or phenylalanine (D816F). Dasatinib inhibited the autophosphorylation of human KIT D816V and D816F with an IC50 of ∼100 nmol/L. However, the IC50 for inhibition of autophosphorylation of the KIT D816Y mutation was significantly lower (IC50 1-10 nmol/L; Fig. 4A ).
In comparison, imatinib in doses of up to 10,000 nmol/L did not significantly inhibit KIT autophosphorylation in D816V and D816F cells. However, D816Y cells were moderately sensitive to imatinib therapy with doses of >1,000 nmol/L but <10,000 nmol/L completely inhibit KIT autophosphorylation ( Fig. 4B). These findings are in agreement with our results for the murine p815 (D814Y) cell line shown in Fig. 3D-F.
Dasatinib is 1 log more potent against WT or juxtamembrane domain mutant KIT isoforms than activation loop mutant KIT isoforms. Our results suggested that dasatinib was less active against KIT activation loop mutant isoforms than against WT or juxtamembrane domain mutant KIT. However, we were unable to directly compare this in our isogenic Ba/F3 system, as we could not establish an IL-3-independent cell line expressing WT or juxtamembrane domain mutant KIT. Thus, we could not directly compare WT KIT to activation loop mutant KIT in the same cellular context. Therefore, we did additional experiments in which we transiently transfected CHO-K1 cells with expression vectors encoding WT or mutant KIT isoforms. Transfected cells were treated with dasatinib and biochemically analyzed as described above. Consistent with our previous results, dasatinib inhibited the autophosphorylation of SCF-stimulated WT KIT (analogous to M-07eE) or juxtamembrane domain mutant KIT (analogous to the mutation in HMC-1.1) with an IC50 of 1 to 10 nmol/L, whereas the IC50 for inhibition of autophosphorylation of the KIT D816V and D816H mutations [reported in <5% of systemic mastocytosis ( 33) and 7% of seminoma cases ( 8)] was ∼100 to 500 nmol/L ( Fig. 4C). Some lane-to-lane variation in total KIT expression is apparent in Fig. 4; such differences are partially attributable to variations in efficiency of immunoprecipitation between experimental conditions and also to the previously described inverse association between kinase activation and protein degradation (i.e., KIT turnover is increased when the kinase is activated; ref. 34).
Dasatinib inhibits KIT-dependent activation of downstream signaling pathways. We studied the effects of inhibition of KIT kinase by dasatinib on the activation status of KIT-dependent downstream signaling pathways, including MAPK, AKT, and STAT3. Figure 5 shows representative Western blots for factor-independent Ba/F3 cells expressing human KIT D816V, D816F, or D816Y mutations. For comparison, we also analyzed the effects of dasatinib of activation of MAPK1/2, AKT, and STAT3 in HMC-1.1, HMC-1.2, and p815 cells. MAPK1/2 (ERK1/2), STAT3, and AKT are constitutively activated in these cells. The phosphorylation of STAT3 and MAPK1 was potently and completely inhibited in dasatinib-treated BaF3 D816V/Y/F cells with IC50 values that were similar to those required for inhibition of KIT autophosphorylation. STAT3 activation was potently but incompletely inhibited in dasatinib-treated p815 and HMC-1.2 cells. In contrast to the other cell lines, STAT3 activation was only minimally inhibited in dasatinib-treated HMC-1.1 cells. AKT activation was potently but incompletely inhibited in all tested cell lines using dasatinib doses of 10 to 1,000 nmol/L. Similarly, MAPK2 activation was also less potently inhibited than MAPK1 ( Fig. 5). Notably, dasatinib-induced inhibition of proliferation and induction of apoptosis are strongly correlated with inhibiting the activation of KIT, MAPK (MAPK1 > MAPK2), and AKT. In contrast, STAT3 activation does not correlate with cellular proliferation and/or avoidance of apoptosis.
Effects of dasatinib on cellular proliferation and survival of isogenic cells expressing KIT D816 F/V/Y. Consistent with the results of our biochemical studies, dasatinib inhibited the proliferation of Ba/F3 KIT D816V and D816F cells with an IC50 of 100 to 150 nmol/L, and Ba/F3 KIT D816Y cells with an IC50 of 5 nmol/L. In contrast, imatinib had no significant inhibitory effect on the growth of these three cell lines (IC50 > 10,000 nmol/L; Fig. 6A ). Dasatinib also potently induced apoptosis of the Ba/F3 KIT D816V and D816F cell lines with calculated IC50 values of 220 and 120 nmol/L, respectively. The IC50 for induction of apoptosis of the Ba/F3 D816Y cell line was 20 nmol/L ( Fig. 6B). Therefore, dasatinib was at least 1 log more potent against KIT D816Y than against KIT D816V/F. Addition of murine IL-3 (5 ng/mL) to Ba/F3 D816V cells prevented dasatinib-induced apoptosis [77% viable cells when cultured with dasatinib 1000 nmol/L + IL-3 versus 0.1% viable cells when cultured in 1,000 nmol/L dasatinib and no IL-3 (data not shown)].
Crystal structures of imatinib bound to the kinase domains of ABL or KIT indicate that imatinib binds to the ATP-binding site of these kinases only when the activation loop of the kinase is in the inactive or “closed” conformation ( 17, 18, 35– 37). One mechanism of acquired resistance to imatinib in CML is the development of mutations of the BCR-ABL activation loop that stabilize the kinase activation loop in the active conformation, thus preventing imatinib binding (e.g., V379I, L387M, and H396R; ref. 38). Structural studies of the pyrido[2,3-d]pyrimidine class of dual SRC-ABL inhibitors show that these compounds also bind to the ATP-binding site in ABL but without regard for the position of the activation loop, which can be in either the active or inactive conformation ( 19, 21, 22, 35). Notably, these compounds can inhibit the kinase activity of certain imatinib-resistant BCR-ABL activation loop mutant isoforms. Based on these observations, Shah et al. ( 20) profiled the activity of dasatinib against a panel of cell lines expressing WT or imatinib-resistant BCR-ABL. Dasatinib inhibited the kinase activity of 14 of 15 imatinib-resistant BCR-ABL mutants, including all tested activation loop mutants. Thus, dasatinib is predicted to bind to the ATP-binding site of BCR-ABL irrespective of the conformation of the activation loop ( 20).
Gain-of-function point mutations of the KIT activation loop are associated with certain human neoplasms, including systemic mast cell disorders ( 13, 33), AML ( 5), seminoma/dysgerminoma ( 8, 39), and GIST (both primary and imatinib-resistant GIST; refs. 12, 40). In the case of mast cell disorders, seminoma, and AML, the most frequent KIT mutation is the replacement of the normal aspartic acid residue at codon 816 of the activation loop with a valine residue (D816V). The D816V mutation results in constitutive activation of KIT kinase activity and is predicted to help stabilize the activation loop in the active conformation. In addition to D816V, other mutations involving codon 816 have been reported in systemic mast cell disorders (D816Y and D816F; refs. 13, 33), AML (D816Y; refs. 5, 41), and/or seminomas (D816Y and D816H; refs. 8, 9, 42). Consistent with the structural model of imatinib binding to KIT, the kinase activity of all of these mutants is resistant to imatinib ( 2, 8, 13).
Based on previous reports of the activity of some small-molecule compounds against KIT activation loop mutations ( 21, 43) and specifically, of some SRC and/or ABL inhibitors against KIT ( 19), we hypothesized that dasatinib might also inhibit the kinase activity of KIT. In our studies, we found dasatinib to be a potent inhibitor of WT KIT with an IC50 for inhibition of autophosphorylation and cellular proliferation of 5 to 10 nmol/L. In comparison, the IC50 for inhibition of autophosphorylation and proliferation in these same cells by imatinib was 10- to 20-fold higher (∼100 nmol/L; ref. 11). The IC50 for dasatinib inhibition of KIT autophosphorylation that we obtained using cell-based assays of full-length KIT is very similar to that reported by Lombardo et al. ( 19) using cell-free assays of kinase domain–only recombinant KIT enzyme.
Juxtamembrane domain mutations of KIT are commonly associated with human GISTs (summarized in ref. 12) and a minority of cases of systemic mastocytosis ( 33) and AML ( 5, 44). Dasatinib also potently inhibits KIT juxtamembrane domain mutations with an IC50 of 1 to 10 nmol/L. Notably, dasatinib had similar potency to imatinib for inhibition of KIT autophosphorylation and cellular proliferation in a mast cell line expressing juxtamembrane domain mutant KIT (HMC-1.1) and was even more potent than imatinib for inducing apoptosis of this cell line.
Dasatinib is a much more potent inhibitor of KIT activation loop mutants than imatinib, with IC50 values for inhibition of autophosphorylation of KIT D816 mutants in the range of 10 to 100 nmol/L. Interestingly, the potency of the dasatinib against KIT kinase is differentially influenced by various activation loop mutations. Notably, KIT D816Y is 10-fold more sensitive to dasatinib than KIT D816V/F. In addition, KIT D816F is ∼2-fold more sensitive to dasatinib compared with KIT D816V.
Our results suggest that the conformation of the KIT activation loop does influence dasatinib potency, perhaps due to secondary changes in the ATP-binding pocket that influence drug binding. Alternatively, the different activation loop mutations might have differential abilities to stabilize the activation loop in the active conformation and prevent KIT from assuming an inactive kinase conformation that has higher dasatinib-binding affinity. This hypothesis is supported by our observation that imatinib is more potent against D816Y than against D816V/F. Further structural studies are needed to explain these experimental results and to compare the dasatinib liganded structures of KIT and ABL.
In our studies, inhibition of KIT kinase in HMC-1.1 (human mastocytosis) and p815 (murine mastocytosis) resulted in inhibition of cellular proliferation and induction of apoptosis. This suggests that therapeutic inhibition of KIT kinase would be effective for human mastocytosis that is associated with KIT D816 mutations. There are other lines of evidence to support this hypothesis: (a) KIT kinase inhibition is developmentally required for mast cell formation ( 1, 3, 4); (b) imatinib-induced inhibition of an alternative oncogenic kinase (FIP1L1-PDGFRA) results in marked clinical responses in variant systemic mastocytosis associated with this genomic alteration ( 45); (c) inhibition of KIT D816V by the kinase inhibitor PKC412 resulted in hematologic and clinical improvement in a patient with mast cell leukemia ( 46).
It should be noted that the HMC-1.2 cell line (human mastocytosis with KIT V560G/D816V) was less sensitive to the antiproliferative effects of dasatinib. Dasatinib potently inhibited KIT in these cells but this seemed to be insufficient to inhibit cellular proliferation or induce apoptosis. We speculate that other secondary oncogenic events, which developed during the prolonged cell passaging that gave rise to this cell line, are responsible for the apparent “disconnect” between inhibiting KIT kinase and effects on cellular proliferation and survival.
Dasatinib is currently in phase I/II trials for CML. Based on the preliminary reports of these studies, it seems that this drug is safe, well tolerated, and efficacious in the setting of imatinib-resistant CML ( 47). The pharmacokinetic data from these trials indicate that drug levels required to inhibit KIT activation loop mutations shown in the present studies can be safely achieved in the systemic circulation of patients. Based on our studies, we predict that dasatinib would have biological and clinical activity against human diseases associated with KIT activation loop mutations, including systemic mastocytosis ( 33), AML ( 5, 44), CDDP-resistant/refractory ( 39), seminoma/dysgerminoma ( 8, 9), and imatinib-resistant GIST ( 12).
Grant support: Merit Review grant from the Department of Veterans Affairs (M.C. Heinrich), the Doris Duke Charitable Foundation (M.C. Heinrich), the Deutsche Krebshilfe Foundation (M.M. Schittenhelm), and flow cytometry support from the Flow Cytometry Shared Resource of the Oregon Health and Science University Cancer Institute grant P30 CA69533.
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.
Note: F.Y. Lee is employed by Bristol-Myers Squibb, Inc., whose product was studied in the present work.
- Received June 15, 2005.
- Revision received September 18, 2005.
- Accepted October 26, 2005.
- ©2006 American Association for Cancer Research.