Transcriptional Changes Induced by Imatinib and Nilotinib in the Chronic Myelogenous Leukemia (CML) Cell Line K562.
Authors: Bruennert Daniela , Bruns Ingmar , Gattermann Norbert , Kronenwett Ralf , Haas Rainer , Neumann Frank 
Published: October 2019

Abstract
Abstract 4250
Nilotinib is a selective bcr-abl tyrosine kinase inhibitor that is 30-fold more potent than Imatinib in vitro. To examine the molecular and functional effects of Nilotinib and Imatinib we performed gene expression and functional analyses in K562 cells following in vitro treatment with the two tyrosine kinase inhibitors. Affymetrix U133A 2.0 microarrays covering 21.722 probe sets were used to analyse the gene expression profile of 5×10 7 K562 cells after 24h in vitro treatment with Imatinib (0.5 μM) or Nilotinib (0.05 μM) (half maximal inhibitory concentration). Gene expression data of the treated cells were compared with data of untreated cells. In addition, proliferation (MTS Assay, Promega), apoptosis (Cell Death Detection ELISAPLUS, Roche) and cell cycle (FITC BrdU Flow Kit, BD Pharmingen) assays were performed. Protein levels of STAT5, pSTAT5(Thr694), PIM-1 and PIM-2 were detected with Western blots. Particular emphasis was put on 303 genes which we found to be differentially expressed in primary CD34+ cells from patients with CML in vivo during treatment with Imatinib (Bruennert et al, Leukemia, 2008). In K562 cells, we found that Imatinib led to a significant differential expression of 45 of those genes. In general, the effect of Nilotinib with regard to the number of genes affected and degree of suppression was more pronounced resulting in a significant differential expression of 120 genes of the aforementioned genes. Of note, genes affected by Nilotinib included all genes altered by Imatinib. Downregulation of genes involved in cell cycle was observed in 17 genes following Nilotinib exposure, but only in the PIM-1 gene following Imatinib exposure. This effect of Nilotinib is in line with the results of cell cycle experiments showing that Nilotinib exposed cells had the lowest proportion of actively cycling cells. The proportion of apoptotic K562 cells was 5.5-fold greater following treatment with Nilotinib in comparison to treatment with DMSO, whereas Imatinib treated K562 cells had a 1.3-fold higher apoptosis rate compared to DMSO treated 562 cells after 24 hours. The superiority of Nilotinib is also reflected by the results of protein analysis of STAT5, pSTAT5(Thr694), PIM-1 and PIM-2: Both tyrosine kinase inhibitors completely inhibit the phosphorylation of STAT5, but Nilotinib inhibits PIM-1 and PIM-2 stronger than Imatinib (by 85% vs. 70%) in K562 cells. In summary, on a molecular level Nilotinib is apparently more potent than Imatinib with regard to the number of genes affected and the degree of their suppression. Among the 45 genes that were significant differentially expressed with both drugs, genes of Imatinib treated K562 cells were downregulated 1.68fold (mean) whereas genes of Nilotinib treated K562 cells were downregulated 2.41fold (mean). The genes altered are mainly associated with cell cycle regulation. In addition to this Nilotinib has a stronger inhibitory effect on PIM 1 and 2.

Disclosures:
Gattermann: Novartis: Honoraria, Participation in Advisory Boards on deferasirox clinical trials.