Changes associated with the development of resistance to imatinib (STI571) in two leukemia cell lines expressing p210...

Although various mechanisms have been recognized as being associated with the development of resistance to imatinib mesylate in vitro and in clinical situations, their relative significance and contributions remain poorly understood, as is the sequence of events leading to the selection of the resistant phenotype. Experimental in vitro systems involving well defined cell lines and conditions can be used to some advantage to answer specific questions and to develop in vitro models of imatinib resistance that would reflect its potential heterogeneity.

Two cell lines, KBM5 and KBM7, which expressed p210 Bcr/Abl and which differed in their inherent sensitivity to imatinib, the number of copies of the BCR/ABL fusion gene, and the activation of apoptotic pathways, were grown in vitro in the presence of increasing concentrations of imatinib. The resistant cells were analyzed for cell cycle progression, apoptotic response after exposure to imatinib, expression of Bcr/Abl, tyrosine kinase activity, and the presence of mutations within the adenosine triphosphate (ATP) coding domain of BCR/ABL. At various levels of resistance, the cells were transferred into drug-free media, and the stability of the resistant phenotype was determined in the absence of the drug.

In KBM7 cells, the development of resistance was characterized by loss of apoptotic response to the drug, amplification of BCR/ABL, increased levels of expression of p210 Bcr/Abl, and decreased inhibition of Bcr/Abl tyrosine kinase (TK) activity by imatinib. No mutations within the ATP-binding domain of Bcr/Abl were identified, and resistance remained stable in the absence of the drug. In KBM5 cells, which previously were found to be characterized by the acquisition of a single C-T mutation at ABL nucleotide 944 (T315I) at high levels of resistance, this same mutation was detected at an intermediate level, but not at a low level, of resistance. The response of KBM5 cells to imatinib was characterized by a low level of apoptotic response, a marginal increase in BCR/ABL copy number, a modest increase in p210 expression, and a highly imatinib-resistant Bcr/Abl TK. Partial reversal of resistance was observed in highly resistant KBM5-STI571(R1.0) cells, which continued to display the C-T mutation. In KBM5 cells with an intermediate level of resistance, the T315I mutation was no longer detectable upon their reversal to the sensitive phenotype.

BCR/ABL amplification with subsequent overexpression of Bcr/Abl protein, loss of apoptotic response, or point mutation of the ATP-binding site of BCR/ABL was associated alternatively with the acquisition of the resistant phenotype, supporting the notion that multiple mechanisms are involved in the induction of resistance to imatinib. The initial number of BCR/ABL copies itself was not related directly to the degree of resistance. The reversibility of the resistance may be complete, partial, or irreversible, depending on the mechanism(s) involved and the degree of resistance. Both cell lines serve as models for further elucidation of various aspects of imatinib-resistance mechanisms.