Le Coutre et al1 report the characterization of a cell line (LAMA84R) that expresses the oncogenic tyrosine kinase Bcr/Abl but is resistant to the tyrosine kinase inhibitor STI571. Those authors argue that this resistance is solely due to overexpression of the Bcr/Abl protein as a result of gene amplification.
An alternative explanation for this data is that some fraction of the Bcr/Abl in the resistant cell line has developed partial resistance to STI571 through a point mutation in the ATP binding region. To test this hypothesis, le Coutre et al amplified a BCR/ABL fragment from both cell lines, sequenced the product, and compared it to the known nucleotide sequence. For this comparison, those authors chose a stretch of 87 nucleotides (29 amino acids1(Figure 5)) that they refer to as the “ATP binding domain” of BCR/ABL. How this stretch of sequence was chosen is unclear, but what is certain is that it does not represent even the major fraction of the ATP binding region of BCR/ABL. The residues that directly contact ATP in a typical protein kinase span a region of 130 to 160 amino acids in primary sequence. Even this subset does not include many residues that do not directly contact ATP, but which when mutated could readily have structural effects that influence inhibitor sensitivity.
Resistance of Bcr/Abl to STI571 would presumably be indicated by reduced sensitivity to the drug in an in vitro kinase assay. Le Coutre et al found that both cell lines display sensitivity to STI571 but that “[d]iffering from Western blot experiments, the inhibition of kinase activity [in vitro] was never complete, even at 10 μM [concentration of STI571].” 1(p1762) This result is inconsistent with the reported potency of STI571 against the Abl kinase in vitro (IC50 = 0.038 μM)2 and suggests that some fraction of the immunoprecipitated Abl kinase activity in this study possessed reduced sensitivity to the drug.
Le Coutre et al attempt to reconcile this discrepancy in the following way: “However, it has to be considered, in the immunocomplex kinase assay, that very high, nonphysiological ATP concentrations (>20 μM) are present, which compete with STI571 for the ATP-binding pocket of bcr/abl.” 1(p1762)This statement is not true. Physiological ATP concentrations typically range from 1 to 10 mM, which is 100-fold higher than le Coutre et al's reported assay conditions; as a competitive binder, STI571 should actually be more potent against the immunocomplexed kinase than in vivo.3
A thorough characterization of mechanisms of cellular resistance to STI571 would provide valuable insight into the potential utility of competitive kinase inhibitors as therapeutics. The present study, however, presents incomplete evidence that cannot be consistently explained by the hypothesis that resistance is solely due to gene amplification.
Gene amplification the most likely mechanism of resistance to STI571 in LAMA84R cells
In our recent publication (le Coutre et al1-1), we presented the generation and analysis of a cell line positive for BCR/ABL but resistant to the tyrosine kinase inhibitor STI571. The cell line generated in our laboratory (LAMA84R) exhibited a 10-fold decrease in sensitivity to STI571, which normally induces apoptosis in sensitive parental LAMA84 cells.1-2 Our results indicated that the induction of resistance in LAMA84R is likely to be caused by the amplification of the BCR/ABL gene leading to increased expression of the oncogenic fusion protein in resistant cells. Our conclusion was based on the observation that the transcription and expression of bcr/abl is increased in LAMA84R compared to parental cells as shown by Northern and Western blot analyses. Using fluorescence in situ hybridization (FISH) analysis, we demonstrated that the alteration of bcr/abl expression was accompanied by gene amplification of BCR/ABL in cells resistant to STI571 resulting in an approximately 4-fold increase in bcr/abl expression.
To investigate the possibility of resistance to STI571 caused by a point mutation within the BCR/ABL gene that could result in a reduction of binding affinity to STI57l, we have performed sequence analysis of the ATP binding region located within the Abl portion of the fusion protein. STI571 binds to bcr/abl as a competitor of ATP.1-3 The complete tyrosine kinase domain of cAbl spans amino acids 235-486 and includes the putative ATP binding lobe composed of the conserved amino acids 248-256 and 271, respectively.1-4 These amino acids are encoded by the corresponding nucleotides 744-813 of the cAbl coding sequence (GenBank accession number M14725; see also SwissProt accession number P00519). The nucleotide sequence of the ATP binding domain and approximate flanking sequences derived from LAMA84R appeared to be identical to the published one. But the possibility that single copies of the amplifiedBCR/ABL genes contain mutations that render the resulting protein less susceptible to STI571 cannot be entirely excluded even by sequencing the entire gene, as proposed by Dr Knight. LAMA84R contain approximately 14 copies of the BCR/ABL gene. Because reverse transcription and subsequent sequencing of the resulting PCR product have to be employed, the exact copy of the BCR/ABL gene being analyzed cannot be determined. It is therefore possible that aBCR/ABL gene not presenting the putative mutations be sequenced, with false negative results.
Following our publication,1-1 2 additional reports (Mahon et al1-5 and Griffin and Weisberg1-6) examined the development of resistance to STI571 in vitro. In these reports upregulation of bcr/abl expression either transiently or by gene amplification were suggested to constitute major mechanisms by which resistant cells overcome the apoptotic effect of STI571. Both groups performed PCR analysis of the BCR/ABL gene. Whereas one group analyzed the nucleotide sequence encoding the entire kinase domain,1-5 the second group performed sequence analysis of a putative ATP binding region that was smaller than the one analyzed in our report.1-6 But in neither publication was the detection of a mutation within the ATP binding region nor kinase domain reported. In addition, one group1-6 also probed the BCR/ABLgene by Southern blot assay, employing several restriction enzymes to look for mutations, with negative results.
The mechanism of resistance to STI571 proposed in our publication is supported by our finding that LAMA84R cells remain susceptible to higher doses of the drug. This was demonstrated by dephosphorylation of bcr/abl detected by antiphosphotyrosine Western blotting and the reduction of kinase activity measured by substrate phosphorylation of GST-CH1-Shc in vitro. In both assays, we observed a parallel reduction of bcr/abl kinase activity in both LAMA84R and nonresistant parental cells.
As Dr Knight correctly points out, physiological ATP concentrations are approximately 100-fold higher than the 20 μM used in immunoprecipitation kinase assay. But whereas the ATP pool inside cells is competed for by many different enzymes, in the kinase assay it is at the complete disposal of bcr/abl. In addition, it must be remembered that the levels of phosphorylation inside cells (as evaluated by Western blot assay) reflect the balance between phosphorylation and dephosphorylation, whereas in the kinase assay no dephosphorylation is possible because phosphatases are not assumed to be present and phosphatase inhibitors are added in the reaction buffer. For all these reasons, as we pointed out, neither of the 2 assays used to analyze the STI571-sensitivity of bcr/abl derived from LAMA84R and parental cells allow the exact quantification of kinase activity but are designed to compare bcr/abl derived from resistant and susceptible cell lines. Band intensity between the 2 assays therefore cannot be compared directly.
Our study provides first evidence that resistance to STI571 can be caused by the amplification of the BCR/ABL gene in vitro. Although it is not completely possible to rule out mutations, we think the most likely explanation for the observed resistance in this case resides in the amplification of the BCR/ABL gene. Other publications that report the generation of similar cell lines and analyze mechanisms of resistance to STI571 complement our original results.
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