Point mutations in the KD of Bcr-Abl that impair drug binding have been established as the major mechanism of acquired resistance to imatinib (1
). Here we provide evidence that, in addition to conferring drug resistance, KD mutations significantly affect the transformation potency of Bcr-Abl. The transformation potency of five common KD mutants compared to native Bcr-Abl was found to depend not only on the type of mutant but also on the specific growth conditions and cell type. For example, the P-loop mutants E255K and Y253F showed a pronounced increase of potency in primary B cells and Ba/F3 cells grown in low serum, while there was no significant difference compared to native Bcr-Abl in myeloid colony formation assays. T315I was equipotent to native Bcr-Abl in Ba/F3 cells, primary B cells, and clonogenic assays performed in the presence of either 10% FBS or growth factors but had a severe transformation defect in the absence of both. This suggests that T315I, unlike native Bcr-Abl, may be unable to activate pathways that compensate for the lack of cytokines and serum. These observations indicate that the competitiveness of a given Bcr-Abl KD mutant is influenced in a complex fashion by intrinsic and extrinsic factors. Based on all in vitro assays, an approximate consensus ranking of transformation potency is Y253F > E255K > native Bcr-Abl ≥ T315I > H396P > M351T (Table ).
In contrast to the in vitro assays, no statistically significant differences were observed in a transduction/transplantation model of CML, although there were trends for longer survival in mice transplanted with BM expressing the H396P mutant and for shorter survival in the recipients of BM transduced with E255K. The lack of significant survival differences may reflect the aggressive nature of the murine CML model that may obscure more subtle effects of the mutations on disease biology. This finding is consistent with the Ba/F3 cell proliferation and the clonogenic assays, where differences were decreased or even abrogated under optimal growth conditions.
To investigate whether differences in intrinsic kinase activity may account for the differences in transformation potency, we determined kinetic parameters in substrate phosphorylation assays. In an initial series of experiments, we used the isolated KDs of Abl. Here, the kcat
value of the Y253F mutant was increased over native Bcr-Abl, the kcat
value of E255K was similar, and the kcat
value of the other mutants were slightly (H396P) or significantly (M351T and T315I) reduced. Analysis of the full-length proteins confirmed the findings for Y253F, T351I, and M351T but in contrast to the isolated KD showed a slight reduction of kcat
value for E255K and a slight increase of kcat
for H396P. The subtle differences between the isolated KD and the full-length Bcr-Abl constructs point to regulatory mechanisms that are present only in the full-length proteins. The results of our in vitro kinase assays are in contrast to data reported by Yamamoto et al., who found that both E255K and T315I show increased kinase activity toward recombinant GST-CrkL relative to native Bcr-Abl (39
). However, there are several factors in their experiments that would not allow precise determinations of kinase activity. The constructs used contained only a small fragment of Bcr fused N terminally to the Abl SH3 domain, lacking both the N-terminal “cap” region of Abl, critical to the autoinhibition of the kinase, and the coiled-coil motif of Bcr that mediates dimerization of Bcr-Abl (23
). Thus, the mechanism of kinase activation in these constructs may differ from full-length Bcr-Abl, similar to our experiments using the isolated kinase domains. In addition, anti-Flag immunoprecipitates were used in the kinase assays, which may contain additional proteins that could influence kinase activity. Similar limitations apply to data recently published by another group who failed to detect differences in kinase activity in anti-Abl immunoprecipitates of cell lines expressing T315I and native Bcr-Abl, respectively (19
Autophosphorylation of Abl on tyrosines 226 (phosphorylated in all mutants and native Bcr-Abl) and 393 (phosphorylated in native Bcr-Abl and all mutants except H396P) influence kinase activity as well as imatinib sensitivity (20
). This regulation appears to require full-length protein, as Nagar et al. demonstrated that in the isolated KD of Abl, phosphorylation affected imatinib binding but not kinase activity (22
). Using dephosphorylated proteins, Roumiantsev et al. found no difference in kinase activity between native and Y253F mutant c-Abl, although they observed increased levels of phosphotyrosine in Ba/F3 cells expressing Y253F mutant Bcr-Abl compared to native Bcr-Abl, a finding consistent with increased intracellular kinase activity (30
). In contrast to their results, we were unable to document consistent differences in total cellular phosphotyrosine levels between the various mutants and native Bcr-Abl. The reason for this discrepancy is unclear. It is possible that the relatively subtle differences in intrinsic kinase activity may not be detectable on the relatively insensitive total phosphotyrosine blots. In addition, total phosphotyrosine levels reflect additional factors, such as the activity of phosphatases that operate only in intact cells.
Overall, it is evident that the differences in intrinsic kinase activity only partially explain the differences in transformation potency. For example, the E255K mutant exhibits increased transformation potency, although its kinase activity is slightly reduced compared to native Bcr-Abl. Similarly, the T315I mutant is equipotent to native Bcr-Abl in the majority of assays, although it has consistently much lower kinase activity. These data raise the question of additional factors impacting transformation potency. Guided by reproducible differences observed on total phosphotyrosine blots among the mutants tested, we carried out a detailed analysis of protein tyrosine phosphorylation using mass spectroscopy and identified differentially phosphorylated tyrosine residues on several proteins. Importantly, for a subset of these phosphopeptides, differences were confirmed in an independent “targeted” experiment. However, we cannot exclude that some differences would not be reproducible if the entire analysis was repeated, since some phosphorylation sites may be specific to the individual cell lines rather than the Bcr-Abl mutant. Experiments are under way to validate the results in a series of independently derived BaF/3 and 32D cell lines expressing native Bcr-Abl or the various mutants.
Differentially phosphorylated proteins included known substrates of Bcr-Abl such as Cbl, as well as proteins that had not been previously associated with Bcr-Abl-positive leukemia, such as Scap2 (Table ). Phosphorylation of tyrosine 1021 of SHIP1 was detected in Ba/F3 cells expressing the M351T and H396P mutants of Bcr-Abl but not in cells expressing the Y253F, E255K, and T315I mutants or native Bcr-Abl. Immunoblot analysis confirmed this mass spectroscopy finding. In accord with published data (33
), SHIP1 levels were significantly reduced in Ba/F3 cells expressing native Bcr-Abl compared to the parental cells. SHIP1 has diverse functions in hematopoietic cells. Mice with targeted disruption of SHIP1 develop a myeloproliferative syndrome (14
), a finding consistent with a negative regulatory role in hematopoiesis. In Ba/F3 cells transformed by Bcr-Abl, SHIP1 negatively regulates motility. Thus, re-expression of SHIP1 reduces the enhanced migration of these cells, and this is dependent on Y1021, since a Y1021F mutant of SHIP1 failed to inhibit motility (33
). Thus, the phosphorylation of Y1021 in Ba/F3 cells expressing M351T and H396P may attenuate Bcr-Abl-mediated transformation by reducing cell motility. In addition, SHIP1 has been shown to negatively regulate Akt activity in B cells after cross-linking of FcγRIIB1 (2
). It is not known whether these effects are dependent on Y1021, but the reduction in the phosphorylation of Akt on threonine 308 and serine 473 in cells expressing the M351T and H396P mutants suggests that Y1021 may be involved in mediating the negative regulation of Akt. Further analysis of these differences in signaling should allow insight into pathways that enhance or antagonize transformation by Bcr-Abl.
The detection of Bcr-Abl KD mutations prior to commencing therapy with imatinib would be predicted to be primarily dependent on the transformation potency of the mutant. Consistent with this, we detected Y253F (gain of function) but not M351T (loss of function) in an unbiased analysis of imatinib-naive patients (38
). In contrast, the prevalence of a given Bcr-Abl mutant in a patient undergoing therapy with imatinib will be determined by several factors. The primary factor is its degree of drug resistance. However, other factors, such as the levels of Bcr-Abl expression, may also contribute to the prevalence of a given Bcr-Abl mutant. In addition, we showed that several of the mutant clones demonstrate improved survival compared to native Bcr-Abl under suboptimal growth conditions. As such, it is conceivable that mutant clones may gain a survival advantage in certain niches with lower concentrations of growth factors and cytokines.
Clinical observations suggest that Bcr-Abl kinase mutations may have biological significance beyond conferring drug resistance. The expansion of a clone carrying a somatic mutation may have two explanations. First, the mutation may not be causal to the clone's expansion but solely comigrate with it. If this were the case, one would expect to see random somatic mutations in genes other than BCR-ABL
, in addition to KD mutations. Although this has not been exhaustively investigated, we and others (1
) failed to detect mutations in c-Kit and the platelet-derived growth factor receptors in CML patients with primary or acquired resistance to imatinib, arguing against comigration. Second, the mutation may endow the clone with a proliferative advantage over unmutated cells, which would represent a mechanism of disease progression. Our finding that the P-loop mutants E255K and Y253F have increased transformation potency is in accord with the clinical observation that mutations of the P-loop confer a poor prognosis compared to other mutation types, regardless of their imatinib sensitivity (3
In summary, we provide evidence that KD mutations modulate the biology of Bcr-Abl-induced leukemia irrespective of sensitivity to imatinib. Gain-of-function mutants may independently contribute to disease progression, while loss-of function mutants are selected only in the presence of drug. Intrinsic kinase activity, substrate specificity, and extrinsic factors, such as cytokines, ultimately determine the outgrowth of a given mutant. This information, together with the sensitivity of the various mutants to imatinib and other Abl kinase inhibitors, may be useful to rationally design the optimal therapeutic approach for individual patients.