Contributions of MET activation to BCR-ABL1 tyrosine kinase inhibitor resistance in chronic myeloid leukemia cells

Abstract

Resistance to the breakpoint cluster region-abelson 1 (BCR-ABL1) tyrosine kinase inhibitor (TKI) imatinib poses a major problem when treating chronic myeloid leukemia (CML). Imatinib resistance often results from a secondary mutation in BCR-ABL1. However, in the absence of a mutation in BCR-ABL1, the basis of BCR-ABL1-independent resistance must be elucidated. To gain insight into the mechanisms of BCR-ABL1-independent imatinib resistance, we performed an array-based comparative genomic hybridization. We identified various resistance-related genes, and focused on MET. Treatment with a MET inhibitor resensitized K562/IR cells to BCR-ABL1 TKIs. Combined treatment of K562/IR cells with imatinib and a MET inhibitor suppressed extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) activation, but did not affect AKT activation. Our findings implicate the MET/ERK and MET/JNK pathways in conferring resistance to imatinib, providing new insights into the mechanisms of BCR-ABL1 TKI resistance in CML.

Keywords: ERK1/2; JNK; MET; chronic myeloid leukemia; imatinib resistance.

Figure 1. Establishment of K562/IR cells and their growth curves with imatinib treatment

The effect of various BCR-ABL1 TKIs on cell survival/proliferation was determined using the trypan blue dye exclusion assay. (A) K562 and K562/IR cells were exposed to the indicated concentrations of imatinib. After incubation for 1, 2, 3, 4, 5, or 6 days, the number of viable cells was counted by trypan blue staining. These results are representative of 5 independent experiments. *p < 0.01 vs. untreated K562 cells as assessed with Dunnett's test. (B-G) Cell viability of K562/IR cells and their parental cell lines after exposure to different concentrations of (B) imatinib, (C) nilotinib, (D) dasatinib, (E) bafetinib, (F) ponatinib, (G) DCC-2036, (H) GNF-2, and (I) GNF-5 for 72 h. These results are representative of 5 independent experiments. *p < 0.01 vs. untreated K562 cells as assessed with Dunnett's test.

Figure 2. Inhibition of MET eliminates imatinib resistance in K562/IR cells

(A) Expression of c-MET, WNT2, EZH2, and BRAF in parental and K562/IR cells. Genomic DNA was extracted, and c-MET, WNT2, EZH2, and BRAF levels were determined by real time PCR. The results are expressed as the test:control ratio after normalization using GAPDH. The results are representative of 5 independent experiments. *p < 0.01 vs. K562 cells as assessed by Dunnett's test. (B) Western blotting analysis. Samples of total cell lysates were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes, and incubated with primary antibodies against phospho-MET (Tyr1234/1235), phospho-MET (Tyr1349), phospho-BRAF (Ser445), EZH2, β-CATENIN, MET, BRAF, β-ACTIN, and LAMIN and then with a horseradish peroxidase-conjugate as the secondary antibody. (C-F) K562/IR cells were exposed to the indicated concentrations of imatinib, PHA665752, EPZ005687, FH535, or XAV-939. After incubation for 72 h, the number of dead cells was counted by trypan blue staining. The results are representative of 5 independent experiments. *p < 0.01 vs. untreated K562/IR cells (analysis of variance with Dunnett's test).

Figure 3. The MET/ERK and MET/JNK pathways contribute imatinib resistance

(A) The cytoplasmic fractions of cells were extracted and subjected to SDS-PAGE/immunoblotting with anti-phospho-ERK1/2, anti-phospho-AKT, anti-phospho-JNK, anti-phospho-p38 MAPK, anti-phospho-NF-κB p65, anti-phospho-STAT1, anti-phospho-STAT3, anti-phospho-STAT5, anti-ERK1/2, anti-AKT, anti-JNK, anti-p38 MAPK, anti- NF-κB p65, anti-STAT1, anti-STAT3, and anti-STAT5 antibodies. Anti-β-ACTIN antibody was used as internal standards. (B-E) K562/IR cells were exposed to the indicated concentrations of imatinib, U0126, SP600125, or LY294002. After incubation for 72 h, the number of dead cells was counted by trypan blue staining. The results are representative of 5 independent experiments. *p < 0.01 vs. untreated cells as assessed with Dunnett's test.

Figure 4. Effect of MET, ERK2, and JNK1 siRNAs on imatinib resistance

(A) K562/IR cells were treated with MET siRNA, ERK2 siRNA, JNK1 siRNA, or a negative control siRNA for 1 day. Control cells were treated with PBS and cultured in serum-containing medium for 3 days. phospho-MET, anti-phospho-ERK1/2, anti-phospho-JNK, anti-MET, anti-ERK1/2, and anti-JNK antibodies. Anti-β-ACTIN antibody was used as internal standards. (B-E) K562/IR cells were exposed to the indicated concentrations of MET siRNA, ERK siRNA, or JNK siRNA. After incubation for 72 h, the number of dead cells was counted by trypan blue staining. The results are representative of 5 independent experiments. *p < 0.01 vs. untreated K562/IR cells as assessed with Dunnett's test.

Figure 5. MET inhibitor inhibits the ERK and JNK activation, and combined treatment of MET inhibitor and imatinib significantly suppressed tumor growth of K562/IR cells in vivo

(A, B) K562/IR cells were exposed to the indicated concentrations of imatinib or PHA665752. After incubation for 48 h, the cytoplasmic fractions were extracted and then subjected to SDS-PAGE/immunoblotting with anti-phospho-MET, anti-phospho-ABL1, anti-phospho-ERK1/2, anti-phospho-AKT, anti-phospho-JNK, anti-MET, anti-ABL1, anti-ERK1/2, anti-AKT, and anti-JNK antibodies. Anti-β-ACTIN antibody was used as internal standards. (C) K562 or (D) K562/IR xenograft model. On day 0, mice were treated with imatinib and/or PHA665752. Imatinib was administered orally (p.o.) at 100 mg/kg daily over a period of 2 weeks; n = 5 for each group. PHA665752 was administered intraperitoneally (i.p.) at 15 mg/kg daily over a period of 2 weeks; n = 5 for each group. Tumor volumes are presented as means ± S.E.M. *p < 0.01 vs. controls, and ^#p < 0.01; 100 mg/kg imatinib + 15 mg/kg PHA665752 vs. 100 mg/kg imatinib (ANOVA with Dunnett's test).