Establishment Of A STI571 Resistant Cell Line K562/R And Screening For BCR-ABL Selective Inhibitors Overcoming STI571 Resistance

Targeting the tyrosine kinase activity of Bcr-Abl with STI571 is anattractive therapeutic strategy in chronic myelogenous leukemia (CML). Afew CML cell lines and primary progenitors are, however, resistant to thiscompound.The t(9;22)(q34;q11) reciprocal chromosomal translocation occurs innearly all patients with chronic myelogenous leukemia (CML) and inapproximately 25% of adults and 5% of children with acute lymphoblasticleukemia (ALL). This translocation gives origin to a 22q , or Philadelphia(Ph), chromosome that contains a BCR-ABL hybrid gene, the molecularhallmark of CML and of Ph-positive ALL.BCR-ABL encodes an oncogenicfusion protein of 190, 210, or 230 kd, depending on the breakpoint on theBCR gene .The unifying feature of all these Bcr-Abl fusion proteins is theirderegulated protein tyrosine kinase activity. The latter is responsible for thein vitro transformation effect and the in vivo leukemogenic property ofBcr-Abl.Targeting the tyrosine kinase activity of Bcr-Abl is, therefore, anattractive therapeutic strategy in CML or in BCR-ABL-positive ALL.Toward this aim, several inhibitors of tyrosine kinase were recentlydeveloped.Among these molecules, STI571, previously known asCGP57148B, a 2-phenylaminopyrimidine derivative, is among the mostpromising and selective inhibitors of Bcr-Abl tyrosine kinase activity. Itinhibits competitively the binding of adenosine triphosphate (ATP) to thekinase domain of Abl at micromolar concentrations. Most otherserine/threonine and tyrosine kinases are unaffected, but there is asuppressive effect against the Kit and PDGF receptor kinase activities.Most study have reported that STI571 specifically abrogates CMLgranulocyte macrophage-colony-forming unit (GM-CFU) and erythroidburst-forming unit (BFU-e) colony formation over a 2-log dose range, witha maximum differential effect at 1 μmol/L. The inhibitor also suppressesproliferation of most Ph-positive cell lines.Nevertheless, a fewBCR-ABL-positive GM-CFU colonies from peripheral blood or bonemarrow survive in the presence of the compound, and 2 of 10 Ph-positivecell lines were found to be resistant to STI571.The reasons for this reducedsensitivity to STI571 in some cells is unknown, but it is an issue ofconsiderable relevance as this compound enters clinical trials.We investigated the mechanism of this resistance in two human celllines from which sensitive (s) and resistant (r) clones were generated byvarious methods. Although the resistant cells were able to survive in thepresence of STI571, its proliferation was almost close to that of theirsensitive counterparts in the absence of the compound. The concentrationof STI571 needed for a 50% reduction in viable cells after a 3-dayexposure was on average 235 times higher in the resistant (2.35 μmol/L)than in the sensitive (0.01 μmol/L) clones. The mechanism of resistance toSTI571 varied among the cell line. Thus, in K562/R, there wasup-regulation of the Bcr-Abl protein associated with amplification of theBCR-ABL gene. Sequencing of the Abl kinase domain revealed onemutation—K231N. The multidrug resistance P-glycoprotein (Pgp) wasalso overexpressed in K562/R, indicating that at least 2 mechanisms ofresistance operate in this cell line.In conclusion, our data show that resistance to STI571 inBCR-ABL-positive cells is a rare phenomenon and may develop throughmultiple mechanisms, such as Bcr-Abl overexpression, reduction in theuptake of the compound by Pgp overexpression, or possibly by excessivedegradation. We cannot exclude that the acquisition of compensatorymutations in genes other than BCR-ABL also plays a part. It is possible thatthe same or similar mechanisms operate in a few primary CMLprogenitors that escape the in vitro antiproliferative effect of thecompound. Overall, our data suggest that BCR-ABL-positive cells canevade the inhibitory effect of this tyrosine kinase inhibitor by variousmechanisms.