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Cancer Therapy: Preclinical |
Authors' Affiliations: Departments of 1 Pathology, 2 Medicine, and 3 Surgery, Memorial Sloan-Kettering Cancer Center; 4 Developmental Biology Program and 5 Molecular Pharmacology and Chemistry Program, Sloan-Kettering Institute, New York, New York
Requests for reprints: Cristina R. Antonescu, Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: 212-639-5721; Fax: 212-717-3203; E-mail: antonesc{at}mskcc.org.
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Abstract |
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Experimental Design: In vitro drug screening of stable Ba/F3 KIT mutants recapitulating the genotype of imatinib-resistant patients harboring primary and secondary KIT mutations was investigated. Comparison was made to imatinib-sensitive Ba/F3 KIT mutant cells as well as Ba/F3 cells expressing only secondary KIT mutations. The efficacy of drug treatment was evaluated by proliferation and apoptosis assays, in addition to biochemical inhibition of KIT activation.
Results: Sorafenib was potent against all imatinib-resistant Ba/F3 KIT double mutants tested, including the gatekeeper secondary mutation KITWK557-8del/T670I, which was resistant to other kinase inhibitors. Although all three drugs tested decreased cell proliferation and inhibited KIT activation against exon 13 (KITV560del/V654A) and exon 17 (KITV559D/D820Y) double mutants, nilotinib did so at lower concentrations.
Conclusions: Our results emphasize the need for tailored salvage therapy in imatinib-refractory gastrointestinal stromal tumors according to individual molecular mechanisms of resistance. The Ba/F3 KITWK557-8del/T670I cells were sensitive only to sorafenib inhibition, whereas nilotinib was more potent on imatinib-resistant KITV560del/V654A and KITV559D/D820Y mutant cells than dasatinib and sorafenib.
80% of GIST patients (3, 4), which is directly dependent on the presence and genomic location of KIT/PDGFRA mutations. Patients with KIT exon 11 mutations have a partial response rate of 84%, whereas tumors with an exon 9 mutation or wild-type KIT are less likely to respond (5, 6). Although imatinib has improved survival in GIST, complete response is rare. Furthermore, it is now clear that the majority of patients who initially benefit from tyrosine kinase inhibitors eventually become resistant, with a median time to progression on imatinib mesylate of 2 years (7). After an initial response, 46% to 67% of patients will develop imatinib resistance through acquisition of a secondary mutation in the KIT kinase domain (8–10). These secondary mutations tend to be single amino acid substitutions in exon 17 most often, but they also occur in exons 13 and 14 (8). The mechanism for the development of second-site KIT mutations remains unclear, but resistant patients with identifiable secondary mutations have been treated with imatinib longer than resistant patients lacking secondary mutations (median, 27 versus 14.5 months; ref. 8). These findings suggest that clonal selection of existing mutations before imatinib therapy is unlikely to explain acquired resistance. The frequency of secondary mutations is also determined by the location of the primary KIT mutations, with GISTs harboring KIT exon 11 mutations more commonly becoming imatinib resistant due to acquisition of secondary mutations compared with KIT exon 9–mutated GIST. One possible explanation for this difference is that KIT exon 9 mutant GISTs are able to use alternative mechanisms, whereas KIT exon 11 GISTs are more dependent on KIT signaling (10). Polyclonal acquired resistance as a result of multiple secondary KIT mutations in geographically separate metastases has been reported in up to 18% (10–12). Consistent with secondary clonal evolution, the primary mutation is always detectable in each tumor and there is never more than one new mutation in the same sample. These findings suggest that long-term imatinib therapy leads to clonal selection of resistant tumor subclones. RNA interference KIT knockdown experiments revealed that imatinib-resistant cell lines carrying secondary KIT mutations remain dependent on KIT signaling for growth and survival (10). Alternative mechanisms, such as low-level genomic KIT amplifications, play only a minor role in imatinib resistance in GIST (8, 11). The initial success of imatinib as a targeted therapeutic drug and the better understanding of drug-kinase interaction based on crystallographic structure have led to the development of several novel small-molecule compounds that have a variable spectrum of kinase inhibition. As the median survival is only 15 months once imatinib resistance develops (13), it is important to validate novel therapeutic strategies within the specific KIT/PDGFRA genotype that confer imatinib resistance. Some of the alternative agents to imatinib being tested in clinical trials include nilotinib, sorafenib, and dasatinib. The initial results of these trials are still being analyzed, but the efficacy of these drugs might not be clear due to the heterogeneity of imatinib-resistant patients enrolled. Thus, a more systematic in vitro approach of validating individual drug efficacy vis-à-vis specific resistant genotypes might better guide therapy and clinical trial design. These small molecules function as KIT kinase inhibitors, and some in addition inhibit other downstream targets. Nilotinib (AMN107) was designed based on the crystal structures of imatinib and the ABL kinase complex. This multitargeted kinase inhibitor features higher affinity to the ATP-binding site of ABL kinase to override imatinib resistance caused by BCR-ABL mutation (14). In addition, nilotinib selectively inhibits the KIT and PDGFR tyrosine kinases (14). Another small molecule, which showed great promise in the chronic myelogenous leukemia patients that are resistant or intolerant to imatinib, is dasatinib (BMS-354825). Dasatinib is an ATP competitor, with dual SRC/ABL activity, which inhibits most BCR-ABL mutants by binding to the active form of the kinase (15). Furthermore, dasatinib potently inhibits KIT, both wild-type and juxtamembrane domain mutant KIT (16).
Sorafenib (BAY 43-9006, Nexavar) is a novel biaryl urea compound that was initially developed as a specific inhibitor of serine-threonine kinase RAF. In addition, sorafenib has activity with few receptor tyrosine kinases, including vascular endothelial growth factor receptor-2, vascular endothelial growth factor receptor-3, PDGFRB, and KIT (17). Sorafenib has shown broad-spectrum, dose-dependent antitumor activity against xenograft models of human colon, lung, breast, ovarian, and pancreatic cancers and melanoma (18).
Based on our hypothesis that individual KIT mutant oncoproteins will affect on drug sensitivity, we investigated the efficacy of sorafenib, nilotinib, and dasatinib on Ba/F3 cells expressing various KIT mutants, which recapitulate imatinib-resistant GIST genotypes.
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Materials and Methods |
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The parental cell line Ba/F3 (German Collection of Microorganisms and Cell Cultures, Human and Animal Cell Cultures Collection) is an interleukin-3 (IL-3)-dependent murine pro-B-cell line lacking intrinsic KIT expression. Retroviral vector plasmids containing mutant KIT cDNA isoforms (25 µg of each construct) and the hygromycin selection marker were cotransfected into Ba/F3 cells at the ratio of 25:1 via electroporation using GenePulser (Bio-Rad). Stable transfectants were established by triple selection. The electroporated cells were first selected with hygromycin for 7 to 10 days, and the remaining cells were sorted according to green fluorescent protein fluorescence. The selected green fluorescent protein–positive cells were grown for 2 weeks in the presence of hygromycin, and finally, cells were grown in the absence of IL-3. The cell surface KIT expression was confirmed by flow cytometry using PE-conjugated anti-CD117 (BD Biosciences) antibody. Total KIT expression was confirmed by Western blotting using anti-KIT (Oncogene) antibody.
Cell proliferation assays. To determine the growth inhibition drug effects, Ba/F3 KIT mutant cells were starved from growth factor for 4 h. Fifteen minutes before drug administration, growth factors were added: 10 ng/mL IL-3 for the IL-3–dependent Ba/F3 KIT mutants (KITD820Y and KITV560del/N822K) and 20 ng/mL KL for the remaining KL-dependent Ba/F3 KIT mutant isoforms. Cells were incubated with 10 to 10,000 nmol/L of sorafenib, dasatinib, nilotinib, and imatinib at 37°C for 48 h. The concentrations of drug used in these studies are in line with the concentrations achievable in human plasma based on pharmacokinetic studies of the kinase inhibitors. Dasatinib and nilotinib were synthesized and kindly provided by Dr. Bayard Clarkson (Sloan-Kettering Institute, New York, NY). Imatinib and sorafenib were purchased commercially. Bromodeoxyuridine was then added and incubated for 6 h before harvesting. Cells were fixed and stained using the standard protocol of the BrdU-APC kit (PharMingen). Bromodeoxyuridine incorporation was determined by flow cytometry. All experiments were done in triplicate. A minimum of 2 x 105 events was acquired and the data were analyzed using FlowJo software (version 5.7.2). Cell growth inhibition curves and IC50 values were plotted and calculated using GraphPad Prism software, version 4.03.
Apoptosis assays. Induction of apoptosis on Ba/F3 KIT transformants was evaluated by flow cytometry using the Annexin V-PE Apoptosis Detection kit (PharMingen) in duplicate experiments. Cells at a density of 1 x 106, activated with KL, were cultured in 24-well plates and starved from growth factors for 4 h before drug delivery. Fifteen minutes before adding the drug, growth factors were added. After drug incubation for 48 h using the concentrations indicated above, cells were harvested and stained with anti-Annexin V-PE antibody using the provider's protocol. A minimum of 10,000 events was analyzed by FACScan (Becton Dickinson) within an hour after staining. The data and results were analyzed by FlowJo.
Immunoprecipitation and Western blotting. The Ba/F3 cells expressing single and double KIT mutants were treated with the indicated doses of each drug. Cells were starved from serum and growth factors for 2 h, and only growth factors were added back to medium 15 min before drug administration. Drugs were incubated at 37°C in the absence of serum for 90 min. After treatment, cells were harvested and subjected to protein extraction. Whole-cell lysate (200 µg) was incubated with 2 µg of anti-KIT antibody (Assay Designs, Inc.) for an hour, and then the mixture was incubated with Magna beads (Pierce Biotechnology) overnight at 4°C. The beads were washed and isolated using a magnetic column and resuspended in LDS sample buffer (Invitrogen). Electrophoresis and immunoblotting were done on the protein extracts using the standard protocol. Phosphorylated KIT was detected with anti-phosphotyrosine antibodies PY20 and PY99 (Santa Cruz Biotechnology), and total KIT was detected by mouse monoclonal anti-c-KIT (Santa Cruz Biotechnology). The secondary antibodies used were donkey anti-mouse secondary (Santa Cruz Biotechnology).
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Results |
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Dasatinib is more potent than other kinase inhibitors on imatinib-sensitive Ba/F3 KIT mutants. The in vitro cell proliferation assay showed that dasatinib inhibited cell growth of imatinib-sensitive Ba/F3 KITV559D with an IC50 of 27 nmol/L, whereas imatinib, nilotinib, and sorafenib had an IC50 of 63, 44, and 66 nmol/L, respectively. Dasatinib results are summarized in Table 1 . Dasatinib induced apoptosis in >20% of cells at 10 nmol/L, whereas the other drugs induced programmed cell death at 100 nmol/L. The biochemical assays showed consistent KITV559D phosphorylation inhibition by all four inhibitors tested, with dasatinib being the most potent. KIT kinase activity was distinctly inhibited using <10 nmol/L dasatinib, whereas imatinib, nilotinib, and sorafenib showed the same results at 100 nmol/L.
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65% and sorafenib induced 35% of programmed cell death at the same concentration. Imatinib did not induce overt apoptosis until 1,000 nmol/L. In keeping with the cell proliferation inhibition and apoptosis results, imatinib did not inhibit KIT phosphorylation at 100 nmol/L but showed distinct inhibition on KIT activity at 1,000 nmol/L.
Imatinib-resistant gatekeeper single KITT670I and double KITWK557-8del/T670I mutants are responsive to sorafenib but resistant to dasatinib and nilotinib. Sorafenib was the only drug that inhibited the cell proliferation of the Ba/F3 KITWK557-8del/T670I transfectant with an IC50 of 1,063 nmol/L and induction of significant programmed cell death between 1,000 and 5,000 nmol/L (Fig. 2
). Sorafenib also showed distinct inhibition of the kinase activity of the KL-dependent double mutant KITWK557-8del/T670I isoform at 1,000 nmol/L. In contrast, imatinib, dasatinib, and nilotinib did not inhibit its phosphorylation even up to 10,000 nmol/L. Similar experiments were done on the Ba/F3 cells expressing the gatekeeper single mutant KITT670I, which showed a comparable drug sensitivity profile. Sorafenib significantly inhibited the kinase activity of this single mutant at 1,000 nmol/L, inhibited cell growth with an IC50 of 918 nmol/L, and induced significant apoptosis between 1,000 and 5,000 nmol/L, whereas imatinib, nilotinib, and dasatinib did not show effects on both biochemical and biological assays (Fig. 2).
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Phosphorylation of imatinib-resistant KITV560del/N822K is inhibited by nilotinib at lower concentrations than dasatinib and sorafenib. The Ba/F3 KITV560del/N822K cells did not achieve IL-3 independence. Biochemically, nilotinib completely inhibited KIT phosphorylation at 100 nmol/L concentration, whereas dasatinib and sorafenib at 1,000 nmol/L (data not shown). Proliferation and apoptosis studies done on these IL-3–dependent cells did not show an effect with any of the drugs tested. This result might be related to other IL-3–dependent signaling pathways not targeted by the drugs tested.
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Discussion |
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As shown by several groups, the location of the primary KIT or PDGFRA mutation directly predicts the clinical response to imatinib in GIST patients. Tumors with an underlying primary KIT mutation in exon 11 represent the subgroup with better response rates than other mutational subtypes (5, 6). Furthermore, this group is also at higher risk to develop secondary mutations compared with KIT exon 9–mutated GISTs (8, 11, 12). This observation further supports that the probability of developing a secondary mutation increases with duration of imatinib treatment, which often is longer in GISTs harboring exon 11 mutation than in those with exon 9 mutation or wild-type GISTs. However, the presence of secondary KIT mutations per se within the acquired imatinib resistance setting is not an indicator of a worse prognosis compared with imatinib-refractory tumors without secondary mutations. The latter group escapes the inhibitory efficacy of imatinib through yet unresolved molecular mechanisms (12).
Another level of complexity relies on the fact that long-term imatinib therapy leads to clonal selection of distinct resistant tumor subclones, the so-called "polyclonal acquired resistance" (10–12). Thus, each tumor nodule under progression may undergo individual clonal evolution, resulting in multiple secondary mutations developed at different metastatic sites within the same patient. As such, designing salvage strategies in these imatinib-resistant settings should address inhibition of all the known genomic activating mutations in the oncoprotein.
Most of the prior in vitro drug screening studies on imatinib-resistant KIT mutants investigate only one drug at a time (9, 20; see Table 2 ). Thus, comparing results between different laboratories, using different cell systems (primary GIST cells, Ba/F3, HEK, etc.), is quite difficult. Our study undertakes a detailed investigation of three imatinib alternative small molecules on a large panel of Ba/F3 KIT single and double mutant cells using biological and biochemical methods of assessing drug response.
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Dasatinib is a potent, orally bioavailable inhibitor of several kinases, including BCR-ABL and SRC family of kinases (24). It was recently approved by the Food and Drug Administration for the treatment of chronic myelogenous leukemia and Philadelphia chromosome–positive ALL with resistance or intolerance to previous therapy, including imatinib (25). Dasatinib was previously shown to be more potent than imatinib in inhibiting KITV559D (23), KITV560G (16), and wild-type KIT (15). Our results also indicate that dasatinib is more effective than other kinase inhibitors on imatinib-sensitive exon 11 mutant Ba/F3 KITV559D and KITWK557-8del cells.
Schittenhelm et al. (16) reported that dasatinib inhibits the kinase activity of KITV560G/D816V double mutant cells in a dose-dependent manner. Screening the sensitivity of different activating tyrosine kinase oncoproteins carrying the gatekeeper mutation, Carter et al. (23) showed that dasatinib has no effect on KITV559D/T670I double mutant. Their results also indicate that similar gatekeeper mutations in BCR-ABL (T315I) and EGFR (T670M) oncoproteins are resistant to dasatinib therapy. Our data support that KITT670I expressed either as a single or double mutant oncoprotein in the Ba/F3 cells confers resistance to dasatinib. In contrast, Ba/F3 cells expressing KIT exon 13 or exon 17 single or double mutants were sensitive to dasatinib inhibition. Dasatinib was recently examined in a phase I study in which GIST patients were enrolled (Clinicaltrials.gov identifier NCT00099606); final data are not yet published.
Nilotinib was specifically designed as a more potent inhibitor of mutant and wild-type BCR-ABL compared with imatinib (14). In addition, Weisberg et al. (14) showed that nilotinib is as effective as imatinib in inhibiting exon 11 KITV560del and exon 13 KITK642E mutant Ba/F3 cells. In a different study, nilotinib showed potent inhibition against KITV560G mutant cells (26). Not previously reported, our results indicate nilotinib efficacy on the KIT double mutants involving exon 11 and exons 13 or 17. Specifically, nilotinib induced cell growth inhibition in imatinib-resistant KITV560del/V654A and KITV559D/D820Y mutant cells at lower concentrations than dasatinib and sorafenib. In contrast, nilotinib was not effective against the gatekeeper KIT exon 14 single (KITT670I) or double (KITWK557-8del/T670I) mutant. Similarly, Weisberg et al. (14) showed that, although nilotinib inhibited all the other secondary mutations of BCR-ABL, it had no effect in inhibiting the T315I gatekeeper mutation, which is analogous to the T670I mutation in KIT. Nilotinib was investigated in combination with imatinib in patients who are imatinib resistant (Clinicaltrials.gov identifier NCT00135005); final results of the study are pending.
Previous preclinical data using the Ba/F3 cellular system showed that imatinib-resistant KIT V654A and T670I mutants are sensitive to sunitinib (SU11248; ref. 20). The growth of Ba/F3 cells expressing KITWK557-8del/V654A or KITWK557-8del/T670I was completely inhibited by sunitinib at concentrations below 500 nmol/L (IC50, 50-100 nmol/L; Table 2). Although other imatinib-resistant KIT mutants, such as the more common second kinase domain KIT mutations, were not investigated, Ba/F3 cells expressing PDGFRA-D842V were less sensitive to sunitinib, and their growth proliferation was inhibited at 5 µmol/L.
Anecdotal evidence based mainly on molecular modeling of mutated KIT receptors suggests that some secondary mutations acquired in the imatinib resistance setting might be weaker than others (27). Although most second-site KIT mutations affect the imatinib-binding site, the extent of structural changes conferred varies with the individual amino acid substitution; thus, T670I substantially modifies the binding pocket, whereas V654A induces only relatively confined changes (8, 27). When compared with the biochemical activity of KIT kinase, Tamborini et al. (27) showed that, in both substitutions, the phosphorylation of the receptor persisted in the presence of 3 mmol/L imatinib, in keeping with an activated status of the KIT receptors. However, the single and double mutant KITV654A oncoprotein expressed in COS cells seemed to be unphosphorylated at 6 mmol/L imatinib, whereas the single and double mutant KITT670I did not. These results suggest that the effects of V654A mutation might be subverted by a dose escalation of the imatinib and it would be expected that a clinical response would be restored. Our results are in support of these findings, showing that imatinib partially inhibited the phosphorylation of KITV560del/V654A oncoprotein at 1,000 nmol/L, which was completely inhibited at 5,000 nmol/L. Similarly, induction of apoptosis with imatinib was evident at 1,000 nmol/L in 50% of cells, whereas at 5,000 nmol/L most cells entered programmed cell death.
Alternative salvage options other than inhibiting KIT signaling pathway are presently being explored. These strategies promise a therapeutic solution to the challenge of heterogeneous imatinib resistance. Bauer et al. (28) investigated the activity of heat shock protein 90 inhibitor 17-allylamino-18-demethoxy-geldanamycin (17-AAG) in enhancing cellular degradation of constitutively activated KIT oncoprotein. Thus, 17-AAG was effective in inhibiting KIT-dependent imatinib-sensitive and imatinib-resistant GIST cell lines but not KIT-independent GIST cells, suggesting that its effects result mainly from inactivation of KIT oncoprotein. Taking a different approach, Sambol et al. (29) showed the efficacy of flavopiridol, a cyclin-dependent kinase inhibitor, on a primary GIST cell line carrying an imatinib-sensitive KITK642E exon 13 mutation, by induction of apoptosis and transcriptional down-regulation of KIT. A possible combination of targeted kinase inhibitors with drugs such as flavopiridol or 17-AAG may be more effective in the setting of polyclonal acquired resistance.
In conclusion, second-line tyrosine kinase inhibitors are selectively potent against imatinib-resistant KIT mutations and might provide an effective alternative to imatinib in the management of these patients. Specifically, sorafenib was found to be effective against all the KIT double mutants tested, including the secondary mutation in the "gatekeeper" residue (KITT670I), which is resistant to the other kinase inhibitors. In contrast, imatinib-resistant KITV560del/V654A and KITV559D/D820Y mutant cells were inhibited by all three inhibitors tested, with nilotinib showing a lower IC50 than dasatinib and sorafenib. These in vitro data set the stage for designing future clinical trials, where selection of the second-generation kinase inhibitor will be tailored based on the individual mechanism of imatinib resistance. The presence of a KITT670I gatekeeper mutation for example in the resistant disease may indicate sorafenib or sunitinib as the next therapeutic option, whereas other secondary mutations may be more susceptible to nilotinib.
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Footnotes |
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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.
Received 2/27/07; revised 5/15/07; accepted 6/12/07.
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