Genome‑wide expression and methylation analyses reveal aberrant cell adhesion signaling in tyrosine kinase inhibitor‑resistant CML cells

Abstract

Although chronic myeloid leukemia (CML) can be effectively treated using BCR-ABL1 kinase inhibitors, resistance due to kinase alterations or to BCR-ABL1 independent mechanisms remain a therapeutic challenge. For the latter, the underlying mechanisms are widely discussed; for instance, gene expression changes, epigenetic factors and alternative signaling pathway activation. In the present study, in vitro-CML cell models of resistance against the tyrosine kinase inhibitors (TKIs) imatinib (0.5 and 2 µM) and nilotinib (0.1 µM) with biological replicates were generated to identify novel mechanisms of resistance. Subsequently, genome-wide mRNA expression and DNA methylation were analyzed. While mRNA expression patterns differed largely between biological replicates, there was an overlap of 71 genes differentially expressed between cells resistant against imatinib or nilotinib. Moreover, all TKI resistant cell lines demonstrated a slight hypermethylation compared with native cells. In a combined analysis of 151 genes differentially expressed in the biological replicates of imatinib resistance, cell adhesion signaling, in particular the cellular matrix protein fibronectin 1 (FN1), was significantly dysregulated. This gene was also downregulated in nilotinib resistance. Further analyses showed significant FN1-downregulation in imatinib resistance on mRNA (P<0.001) and protein level (P<0.001). SiRNA-mediated FN1-knockdown in native cells reduced cell adhesion (P=0.02), decreased imatinib susceptibility visible by higher Ki-67 expression (1.5-fold, P=0.04) and increased cell number (1.5-fold, P=0.03). Vice versa, recovery of FN1-expression in imatinib resistant cells was sufficient to partially restore the response to imatinib. Overall, these results suggested a role of cell adhesion signaling and fibronectin 1 in TKI resistant CML and a potential target for novel strategies in treatment of resistant CML.

Clonal evolution in tyrosine kinase inhibitor-resistance: lessons from in vitro-models

“…Three clusters were revealed for the resistant cell lines and 14 clusters for gene sets with highIM-R1, -R3 and -R4 being a distinct cluster separate from the other tested resistant sublines ( Figure 2A ). To compare the network propagation with gene expression data, genome-wide expression analyses of the TKI-resistant cell lines and gene set variation analyses were performed [( 16 ), Figure 2B ]. The resulting pattern of enriched pathways was highly similar to one of the protein-protein interaction network derived from the mutational pattern ( Figure 2A ].…”

“…K-562 cells (RRID: CVCL_0004), established from the pleural effusion of a 53-year-old woman ( 14 ), were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). Cell maintenance, generation of biological replicates of TKI-resistant sublines, and analyses of cell line authenticity were described elsewhere ( 15 , 16 ). Cells were resistant against lowIM (0.5 µM imatinib), highIM (2 µM imatinib), lowN (0.05 µM nilotinib) and highN (0.1 µM nilotinib).…”

“…Microarrays were performed using Clariom S Arrays (Affymetrix; Thermo Fisher Scientific) as previously described ( 16 ). Briefly, RNA was isolated using miRVANA microRNA isolation kit (Thermo Fisher), and 100 ng were hybridized onto the arrays according to the manufacturer’s protocol.…”

“…Cellular fitness was analyzed as previously described (16,18,19). Briefly, cell numbers were obtained by trypan blue staining, WST-1 ( S i g m a -A l d r i c h ) , C a s p a s e G l o 9 A s s a y ( P r o m e g a ) , Bromodeoxyuridine proliferation assay (Merck, Darmstadt, Germany), and MKI ELISA Kit (MyBioSource, San Diego, CA, USA) according to the manufacturers' recommendations.…”