BCR-ABL Gene Expression Is Required for Its Mutations in a Novel KCL-22 Cell Culture Model for Acquired Resistance of Chronic Myelogenous Leukemia

Abstract

Acquired resistance through genetic mutations is a common phenomenon in several cancer therapies using molecularly targeted drugs, best exemplified by the BCR-ABL inhibitor imatinib in treating chronic myelogenous leukemia (CML). Overcoming acquired resistance is a daunting therapeutic challenge, and little is known about how these mutations evolve. To facilitate understanding the resistance mechanisms, we developed a novel culture model for CML acquired resistance in which the CML cell line KCL-22, following initial response to imatinib, develops resistant T315I BCR-ABL mutation. We demonstrate that the emergence of BCR-ABL mutations do not require preexisting BCR-ABL mutations derived from the original patient as the subclones of KCL-22 cells can form various BCR-ABL mutations upon imatinib treatment. BCR-ABL mutation rates vary from cell clone to clone and passages, in contrast to the relatively stable mutation rate of the hypoxanthine-guanine phosphoribosyltransferase gene. Strikingly, development of BCR-ABL mutations depends on its gene expression because BCR-ABL knockdown completely blocks KCL-22 cell relapse on imatinib and acquisition of mutations. We further show that the endogenous BCR-ABL locus has significantly higher mutagenesis potential than the transduced randomly integrated BCR-ABL cDNA. Our study suggests important roles of BCR-ABL gene expression and its native chromosomal locus for acquisition of BCR-ABL mutations and provides a new tool for further studying resistance mechanisms.Chronic myelogenous leukemia results from malignant transformation of a primitive hematopoietic cell by an oncogenic fusion gene BCR-ABL. Treatment with the potent ABL tyrosine kinase inhibitor imatinib mesylate (Gleevec, STI-571, imatinib) results in complete cytogenetic responses in most chronic phase patients with infrequent relapse, but the treatment has poor responses and high frequency of relapse in advanced phases of the disease (1). Clinical resistance to imatinib is mediated primarily by genetic mutations of the BCR-ABL kinase domain and, to a lesser extent, by amplification of BCR-ABL gene (2). Numerous BCR-ABL mutations have been identified in relapsed CML 3 patients, which confer various degrees of resistance to imatinib (2-4). Among them, the T315I mutation is the most resistant in that it does not respond to treatment with the more potent second generation of kinase inhibitors such as nilotinib (5, 6) and dasatinib (7).In contrast to in vivo resistance, nearly all CML cell lines derived from blast crisis CML are sensitive to imatinib treatment in culture (8). Several resistant CML cell lines have been generated by exposing cells to gradually increasing concentrations of imatinib; however, the resulting resistant cells harbor BCR-ABL gene amplification but not mutations (9), in contrast to what is seen in patients. By expressing BCR-ABL cDNA in non-CML cell lines and/or random mutagenesis, multiple studies have demonstrated clinically relevant or novel BCR-ABL mutations that render resistance to imatinib...

De Novo Acquisition of BCR-ABL Mutations for CML Acquired Resistance

“…In contrast to resistance of blast crisis CML in vivo, most blast crisis CML cell lines in culture are very sensitive to 1 M imatinib with exception of KCL-22 (Deininger et al, 1997). Imatinib also kills KCL-22 cells, but it requires higher concentrations and takes longer time (Yuan et al, 2010 highly consistent results among them. This model provides a novel tool for us to look into the process of acquisition of BCR-ABL mutations on the endogenous BCR-ABL in blast crisis CML cells, and has already provided valuable information in our initial study (Yuan et al, 2010) Fig.…”

“…However, mutations in clonal KCL-22 cells may provide a clue. The mutation hot spots are different in three KCL-22 clones (clone L1 for E255K, clone L7 for Y253H and clone Ag 11 for T315I) (Yuan et al, 2010). When the codon changes are examined (TAC to CAC for Y253H, GAG to AAG for E255K, and ACT to ATT for T315I), a common molecular feature for these three mutations can be easily identified, namely, they are all transition mutations.…”

“…KCL-22 cells are positive for myeloid cell markers including myeloid progenitor cell marker CD33. KCL-22 cells harbor two Ph chromosomes but do not have detectable BCR-ABL mutations (Yuan et al, 2010). In contrast to resistance of blast crisis CML in vivo, most blast crisis CML cell lines in culture are very sensitive to 1 M imatinib with exception of KCL-22 (Deininger et al, 1997).…”

“…To address mechanisms of CML acquired resistance, we have recently developed a novel tissue culture model using a naïve blast crisis CML cell line KCL-22 (Yuan et al, 2010). KCL-22 cells were derived from a female blast crisis CML patient, and were characterized as immature undifferentiated cells lacking lymphoid cell characteristics (Kubonishi and Miyoshi, 1983).…”

“…Green for introns, blue for exons, asterisk for T315 mutation, and arrows for PCR primers. Right, relative mutation rate was the ratio of the number of clones bearing mutated endogenous BA or integrated BA cDNA divided by total number of clones, according to Yuan et al, 2010. Although the clinical readout for de novo acquisition and selection of pre-existing mutations are the same, i.e.…”