Induction of resistance to the Abelson inhibitor STI571 in human leukemic cells through gene amplification

Abstract

The 2-phenylaminopyrimidine derivative STI571 has been shown to selectively inhibit the tyrosine kinase domain of the oncogenicbcr/abl fusion protein. The activity of this inhibitor has been demonstrated so far both in vitro with bcr/abl expressing cells derived from leukemic patients, and in vivo on nude mice inoculated with bcr/abl positive cells. Yet, no information is available on whether leukemic cells can develop resistance to bcr/ablinhibition. The human bcr/abl expressing cell line LAMA84 was cultured with increasing concentrations of STI571. After approximately 6 months of culture, a new cell line was obtained and named LAMA84R. This newly selected cell line showed an IC50 for the STI571 (1.0 μM) 10-fold higher than the IC50 (0.1 μM) of the parental sensitive cell line. Treatment with STI571 was shown to increase both the early and late apoptotic fraction in LAMA84 but not in LAMA84R. The induction of apoptosis in LAMA84 was associated with the activation of caspase 3–like activity, which did not develop in the resistant LAMA84R cell line. LAMA84R cells showed increased levels of bcr/abl protein and mRNA when compared to LAMA84 cells. FISH analysis with BCR- and ABL-specific probes in LAMA84R cells revealed the presence of a marker chromosome containing approximately 13 to 14 copies of the BCR/ABL gene. Thus, overexpression of the Bcr/Abl protein mediated through gene amplification is associated with and probably determines resistance of human leukemic cells to STI571 in vitro.

Metabolic characteristics of imatinib resistance in chronic myeloid leukaemia cells

“…Point mutations can directly prevent the binding of imatinib to the Bcr-Abl protein or can lead to conformational changes of the kinase, affecting binding of the inhibitor in an indirect way (Roche-Lestienne et al, 2002). Resistance to imatinib can also be caused by Bcr-Abl overexpression due to amplification of the BCR-ABL gene, as originally described in the LAMA84-r and AR230-r cell lines (le Coutre et al, 2000;Van Etten, 2004). Like most other small molecules, imatinib needs to pass through the cell membrane to reach its target protein.…”

Rational Approaches to the Design of Therapeutics Targeting Molecular Markers: The Case of Chronic Myelogenous Leukemia

“…37 The amplification of BCR-ABL in leukemic cells resistant to imatinib was initially described in cell lines isolated through culture in increasing imatinib concentrations. 38,39 Cells grown in these conditions present with BCR-ABL levels many times higher than those in the original cell lines. The mechanism responsible for BCR-ABL overexpression resides in BCR-ABL amplification, and gene amplification has subsequently been observed in clinical samples obtained from imatinib-resistant pa-tients, with a frequency of around 20% of cases.…”

Kinase-Independent Mechanisms of Resistance of Leukemia Stem Cells to Tyrosine Kinase Inhibitors

“…In fact, the T315I mutation can be detected in some patients even prior to treatment [17]. Other forms of acquired resistance have been described that are independent of mutation in Bcr-Abl but can be attributed to increased expression of efflux and influx proteins [22][23][24], deregulation of apoptosis/survival pathways [25][26][27][28][29][30], or other acquired mutations including amplification of Bcr-Abl [31]. Although this is an interesting and extremely important topic, acquired resistance is not the scope of this article.…”