Blood, 1 December 2000, Vol. 96, No. 12, pp. 4003-4005
CORRESPONDENCE
To the editor:
Another possible mechanism of resistance to STI571
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.
Zachary A. Knight
Department of Cellular and Molecular Pharmacology University
of California, San Francisco San Francisco, CA
References
1.
Le Coutre P, Tassi E, Varella-Garcia M, et al.
Induction of resistance to the Abelson inhibitor STI571 in human leukemic cells through gene amplification.
Blood.
2000;95:1758-1766[Abstract/Free Full Text].
2.
Buchdunger E, Zimmermann J, Mett H, et al.
Inhibition of the abl protein-tyrosine kinase in vitro and in vivo by a 2-phenylaminopyrimidine derivative.
Cancer Res.
1996;56:100-104[Abstract/Free Full Text].
3.
Cohen P.
The development and therapeutic potential of protein kinase inhibitors.
Curr Opin Chem Biol.
1999;3:459-465[Medline]
[Order article via Infotrieve].
Response:
Gene amplification the most likely mechanism of resistance to
STI571 in LAMA84R cells
In our recent publication (le Coutre et al1), 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.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.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.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 amplified
BCR/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 a
BCR/ABL gene not presenting the putative mutations be
sequenced, with false negative results.
Following our publication,1 2 additional reports (Mahon et
al5 and Griffin and Weisberg6) 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,5 the second group performed sequence analysis of a
putative ATP binding region that was smaller than the one analyzed in
our report.6 But in neither publication was the detection
of a mutation within the ATP binding region nor kinase domain reported.
In addition, one group6 also probed the BCR/ABL
gene 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.
Carlo Gambacorti-Passerini, Philipp le Coutre, Elena Tassi, and Holger Ruchatz
Department of Experimental Oncology Istituto Nazionale
Tumori Milan, Italy
References
1.
Le Coutre P, Tassi E, Varella- Garcia M, et al.
Induction of resistance to the Abelson inhibitor STI571 in human leukemic cells through gene amplification.
Blood.
2000;95:1758-1766.
2.
Gambacorti-Passerini C, le Coutre P, Mologni L, et al.
Inhibition of the Abl kinase activity blocks the proliferation of BCR/ABL+ leukemic cells and induces apoptosis.
Blood Cells Mos Dis.
1997;23:380-394[Medline]
[Order article via Infotrieve].
3.
Buchdunger E, Zimmerman J, Mett H, et al.
Inhibition of the Abl protein-tyrosine kinase in vitro and in vivo by a 2-phenylaminopyrimidine derivative.
Cancer Res.
1996;56:100-104.
4.
Fainstein E, Einat M, Gokkel E, et al.
Nucleotide sequcne analysis of human abl and bcr-abl cDNAs.
Oncogene
1989;4:1477-1481[Medline]
[Order article via Infotrieve].
5.
Mahon FX, Deiniger M, Schultheis B, et al.
Selection and characterization of BCR-ABL positive cell lines with differential sensitivity to the tyrosine kinase inhibitor STI571: diverse mechanism of resistance.
Blood.
2000;96:1070-1079[Abstract/Free Full Text].
6.
Weisberg E, Griffin JD.
Mechanism of resistance to the ABL tyrosine kinase inhibitor STI571 in BCR/ABL-transformed hematopoetic cell lines.
Blood.
2000;95:3498-3505[Abstract/Free Full Text].
