Gene expression signatures associated with the in vitro resistance to two tyrosine kinase inhibitors, nilotinib and imatinib

Abstract

The use of selective inhibitors targeting Bcr-Abl kinase is now established as a standard protocol in the treatment of chronic myelogenous leukemia; however, the acquisition of drug resistance is a major obstacle limiting the treatment efficacy. To elucidate the molecular mechanism of drug resistance, we established K562 cell line models resistant to nilotinib and imatinib. Microarray-based transcriptome profiling of resistant cells revealed that nilotinib- and imatinib-resistant cells showed the upregulation of kinase-encoding genes (AURKC, FYN, SYK, BTK and YES1). Among them, the upregulation of AURKC and FYN was observed both in nilotinib- and imatinib-resistant cells irrespective of exposure doses, while SYK, BTK and YES1 showed dose-dependent upregulation of expression. Upregulation of EGF and JAG1 oncogenes as well as genes encoding ATP-dependent drug efflux pump proteins such as ABCB1 was also observed in the resistant cells, which may confer alternative survival benefits. Functional gene set analysis revealed that molecular categories of 'ATPase activity', 'cell adhesion' or 'tyrosine kinase activity' were commonly activated in the resistant clones. Taken together, the transcriptome analysis of tyrosine kinase inhibitors (TKI)-resistant clones provides the insights into the mechanism of drug resistance, which can facilitate the development of an effective screening method as well as therapeutic intervention to deal with TKI resistance.

Figure 1

Unsupervised hierarchical clustering of the 455 genes, which showed differential expression between TKI-resistant K562 sublines and TKI-susceptible parental K562.

Figure 2

Gene expression patterns of 12 gene clusters categorized from the 455 differentially expressed genes.

Figure 3

Gene clusters with transcriptional upregulation in TKI-resistant K562 sublines. (a) Three gene clusters show the upregulation in two nilotinib-resistant sublines as compared with imatinb-resistant or parental K562 sublines. (b) The transcriptional upregulation in both nilotinib- and Imatinib-treated sublines is observed in two gene clusters (Cluster 2 and 9).

Figure 4

Expression levels of five kinases in TKI-resistant K562 sublines. (a) The expression levels of five kinases in four K562 sublines are measured using real-time quantitative PCR. The relative expression levels of AURKC and FYN for three TKI-resistant sublines are illustrated as compared with those of TKI-susceptible parental cell lines. (b) The expression levels of SYK, BTK and YES1 are demonstrated in the same manner.

Figure 5

Expression profiles of the four kinase genes in imatinib-resistant patients and good responders. Twelve CML samples were collected, six poor responders (P) containing three chronic phase (CP) and three acute phase (AP); six good responders (G) containing three CPs and three APs. qRT-PCR was performed for the 12 samples as described in Materials and methods section using the same primers listed in Table 1. All three chronic phase poor responders showed relative upregulation of AURKC expression compared with good responders. However, the other three kinase genes did not show prominent difference of expression between the poor and good responders.