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 pre-existing 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.

FIGURE 1.

Novel model of CML acquired resistance. A, one-half million KCL-22 cells were treated with 1, 2.5, 5, and 10 μm imatinib (STI), and at days after the treatment as indicated, survival cells were counted. Relapse occurred on 2.5 μm and higher concentrations of imatinib 2 weeks post-treatment. B, top, apoptosis of KCL-22 cells after 6 days of treatment with 5 μm imatinib was analyzed by annexin V staining. DAPI, 4′,6-diamidino-2-phenylindole. Bottom, formation of clusters of resistant cells (bright) among scattered dead cells (dark) with 2.5 μm STI-571 treatment for 8 days. C, top, growth curves for resistant cells (KCL-22M) and KCL-22 cells analyzed by XTT. Growth indexes were relative XTT readings normalized to the initial XTT readings at day 0. Bottom, comparison of growth of KCL-22M cells in the absence and presence of imatinib. D, soft agar colony formation of KCL-22 and KCL-22M cells. Five hundred cells were seeded each well in 6-well plates in triplicate. E, comparison of cell size and complexity of KCL-22 and KCL-22M cells. KCL-22M cells exhibited increase at both forward scatter (FS) and side scatter (SC) parameters. SS Lin, side scatter in linear scale. F, comparison of cell cycle of KCL-22 and KCL-22M cells with propidium iodine staining.

FIGURE 2.

Cytogenetic characteristics of KCL-22M cells. A, karyogram of 24-color spectral karyotyping. The composite karyotype is 51,X,del(X)(p11.2p22.3),+der(1;10)(q10;p10),+6,+8,+8,t(9;22) (q34.1;q11.2),der(17;19)(q10;q1),+19,i(21)(q10),+der(22)t(9;22)[10]. B, left, representative three-color FISH of BCR-ABL. Two Philadelphia chromosomes (9/22) were present in all cells. Right, the color code for FISH probes. ASS, argininosuccinate synthetase gene.

FIGURE 3.

Molecular characterizations of the new CML resistance model. A, left, Western blot analysis of BCR-ABL expression and tyrosine phosphorylation in KCL-22 and KCL-22M cells with and without imatinib treatment. The top band (arrow) detected by the tyrosine phosphorylation antibody corresponds to the position of BCR-ABL. Right, immunoprecipitation of KCL-22 cell lysate with BCR-ABL antibody or normal IgG followed by Western analysis for BCR-ABL expression and phosphorylation. P-Tyr, phosphotyrosine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B, sequencing analysis of BCR-ABL kinase domain with cDNA or genomic DNA from KCL-22 and KCL-22M cells. Notice the point mutation of C to T (in bold and red) that causes T315I amino acid change. C, sequencing of BCR-ABL kinase domain mutations in clonal cells. KCL-22M cells derived from 2.5 and 5 μm imatinib treatment were subcloned by limiting dilution, and 10 clones each (liquid culture clones) were sequenced using genomic templates. Clonal cells were separately obtained by plating KCL-22 cells on soft agar with 2.5 or 5 μm imatinib, and 10 colonies each were sequenced. D, mutation frequencies of KCL-22 cells. One million KCL-22 cells were seeded on soft agar with the indicated concentrations of imatinib, and resistant colonies were scored after 3 weeks. E, mutation detection by conventional DNA sequencing. KCL-22 cells cultured without or with 1 μm imatinib for 2 weeks were analyzed. Forty one bacterial clones for untreated cells and 12 for treated cells were sequenced. Mutations were found in only two clones in the latter.

FIGURE 4.

Acquired resistance of clonal CML cells on imatinib treatment. A, time courses of relapse for four KCL-22 clones as analyzed in Fig. 1A. Notice that clones L1 and Ag 11 relapsed on imatinib with time courses similar to parental cells, but clones L7 and Ag3 relapsed with slower time courses. B, resistance of recurrent clonal cells to higher concentrations of imatinib. Recurrent cells derived from 2.5 μm imatinib treatment for four clones were labeled with clone names followed by 2.5R. All recurrent cells were maintained in the medium without imatinib until analysis. Notice the different levels of resistance of L1–2.5R (E255K mutation), L7–2.5R (Y253H mutation), and Ag11–2.5R (T315I mutation) to the higher concentrations of imatinib. Growth of Ag3–2.5R (no mutation) was inhibited by 5 μm imatinib. C, cells from clones L1 and Ag11 were plated on soft agar with 2.5 μm imatinib for 3 weeks, and 10 colonies each were picked for sequencing analysis of BCR-ABL kinase domain mutations. D, mutations from mixed clonal cells. Equal numbers of eight never-relapsed clones were mixed to form a nonrelapse pool (N-pool). Clone L1, L7, or Ag3 was then mixed 1:1 with a nonrelapse pool for resistance analysis in liquid culture, and recurrent cells were analyzed for BCR-ABL mutations.

FIGURE 5.

Comparison of BCR-ABL mutations with spontaneous HPRT mutations. A, measurement of BCR-ABL and HPRT mutation rates. One million cells of each clone were plated on soft agar with 2.5 μm imatinib (STI) or 2.5 μg/ml 6-thioguanine (6-TG) for side-by-side analysis of BCR-ABL and HPRT mutation frequencies, respectively. B, left, BCR-ABL and HPRT mutation rates of KCL-22 cells at passages 5, 16, and 36 measured by soft agar clonogenic assay. Imatinib, 5 μm; 6-thioguanine, 2.5 μg/ml. Right, time courses for relapse of KCL-22 cells on 2.5 μm imatinib at passages 8 and 35. Relapsed cells harbored T315I mutation in both cases. C, HPRT mutation spectrum. Ag11, L1, or KCL-22 cells were seeded on soft agar with 6-thioguanine, and 20 colonies each were picked for analysis of HPRT codon mutations. For Ag11, two colonies had sequencing failure and were excluded.

FIGURE 6.

ROS and DNA damage in KCL-22 cells upon imatinib treatment. A, analysis of ROS in different apoptotic fractions of KCL-22 cells treated with 2.5 μm imatinib for 4 days. ROS increased in all apoptotic fractions of the cells with early apoptotic cells (top left square) having the highest level. Nonapoptotic fraction of the cells (bottom left square) had the lowest level of ROS. DAPI, 4′,6-diamidino-2-phenylindole. B, bulk ROS level decreased in nonapoptotic fraction of KCL-22 cells that were treated with 2.5 μm imatinib for 2 days. Similar results were seen for 1 day of drug treatment (not shown). C, bulk γH2AX level decreased in KCL-22 cells treated with 2.5 μm imatinib for 2 days. D, treatment with anti-oxidants. No effects of NAC or vitamin E were seen to prevent or delay KCL-22 cells from relapse on 2.5 μm imatinib except for 50 mm NAC that itself has significant cytotoxicity (data not shown).

FIGURE 7.

Induction of BCR-ABL mutations is dependent on BCR-ABL expression. A, top, analysis of apoptosis of KCL-22 and KCL-22M cells 6 days after BCR-ABL knockdown. Scrambled shRNA was used for mock knockdown. Less apoptotic cells were noticed in KCL-22M. Bottom, BCR-ABL protein levels after knockdown. DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B, top, effects of BCR-ABL knockdown on acquired resistance on imatinib. After the knockdown, cells regrew, and they were selected for 5 days in puromycin and expanded in drug-free medium. These re-grown BCR-ABL knockdown KCL-22 cells and mock knockdown cells were treated with 5 μm imatinib, and cells were followed as described in Fig. 1. Bottom, BCR-ABL levels in the re-grown knockdown cells. C, effects of BCR-ABL knockdown on formation of imatinib-resistant colonies on soft agar. The re-grown BCR-ABL knockdown (shABL) or mock knockdown KCL-22 cells were plated on soft agar with 5 μm imatinib. The plating efficiency for mock and ABL knockdown was the same (data not shown). D, effects of combining BCR-ABL knockdown with imatinib treatment on acquired resistance. Cells were transduced overnight with lentiviral vectors, and imatinib was added right after the removal of viruses.

FIGURE 8.

Effects of BCR-ABL overexpression on mutations. A, analysis of BCR-ABL expression and total cellular phosphorylation. KCL-22 cells were transduced with empty vector (R1), wild type p210 BCR-ABL (WT), or kinase-inactive p210 mutant (KI). Lysates were collected without or with 2.5 and 5 μm imatinib treatment. B, clonogenic assay of BCR-ABL-overexpressing cells for imatinib resistance with 1,000,000 cells plated each well in triplicate. Imatinib-resistant colonies were plucked and expanded for genomic sequencing of the endogenous BCR-ABL (BA) kinase domain. The number of colonies found with T315I mutation over the total number of colonies sequenced are listed below each category of graph bars. C, analysis of the transduced BCR-ABL cDNA in imatinib-resistant colonies overexpressing wild type BCR-ABL by PCR amplification of 579- and 321-bp fragments of BCR-ABL cDNA from genomic templates using exon primers. Both 579- and 321-bp fragments were sequenced. Only one colony, clone 5 from 2.5 μm imatinib treatment group marked by an asterisk, had BCR-ABL mutation (T315I). Colonies with T315I mutation of the endogenous BCR-ABL were as follows: clones 1, 2, 4, 5, 7, 8, and 10 from 5 μm imatinib group, and clones 1 and 7 from 2.5 μm imatinib group.