MEK 1/2 Inhibitor U0126 Reversed Imatinib Resistance in IM-Resistant K562R
Authors: Gong Yuping , Lin Juan , Niu Ting 
Published: October 2019

Abstract
Abstract 4914

Objective
To study the anti-leukemic activity of MEK inhibitor U0126 alone or in combination with imatinib and explored the reversing mechanism to imatinib resistance in imatinib resistant K562R cell line.

Methods
Cytotoxicity of drug was detected by the MTT assay in the IM-sensitive cell line K562 and IM-resistant cell line K562R. Western Blot assay were employed to examine the expression of p-cAbl, p-Lyn, p-STAT5, ERK signaling pathway (p-cRaf, p-MEK, p-ERK), PI3K/AKT/mTOR signaling pathway(p-AKT, p-mTOR, p-4EBP1) and the apoptosis related proteins (Bax, Bcl2). The apoptosis rates were analyzed by Annexin V/PI double staining flow cytometry assay. The levels of lyn and erk1/2 gene were assayed by RT-PCR.

Results
1. BCR-ABL-independent activation of Lyn and ERK1/2 may be the IM resistant mechanism in K562R cell line.
The IC50 value of K562R cell line(1. 505±0. 459 μmol/L) inhibited by imatinib for 72 hours was higher than the values of K562(0. 159±0. 032μmol/L) and the Resistant Fold(RF)was 9. 465. Western Blot assays showed that p-Lyn, p-MEK and p-ERK of ERK pathway, p-mTOR and p-4EPB1 were over-expressed in K562R cell line relative to K562 cell line. However, the levels of p-cAbl, p-STAT5, p-Raf, p-AKT of PI3K/AKT/mTOR were similar in the K562R and K562 cell lines. The treatment of IM could reduce the expression of p-cAbl and p-MEK, but not that of p-Lyn, p-ERK, p-mTOR, p-4EBP1, and even up-regulate the p-Lyn, p-ERK, p-mTOR. The expression of Bax, Bcl2 and the apoptosis rates were the similar in both cell lines.
2. MEK1/2 inhibitor U0126 could reverse the IM resistance in K562R cell line.
MTT assay showed single-agent U0126 is more sensitive to K562R than K562. The IC50 values of the two cell lines were 34. 235±5. 658 μmol/L and 85. 824±4. 474 μmol/L respectively. The combination of imatinib and U0126 markedly enhanced inhibitory effect as measured by MTT assay in K562R cell line, combination with 10μmol/L U0126, the IC50 values of IM was 0. 134±0. 059μmol/L, which reduced to 8. 9% of single IM treatment.
3. The reversing mechanism of U0126 to imatinib resistance in K562R cell line.
Western Blot showed single IM up-regulated the p-Lyn and p-ERK, while U0126 reduced the expression of them, the combination of the IM and U0126 could synergisticly reduce the p-Lyn expression and neutralize the up-regulation of p-ERK caused by IM single agent. Single IM also up-regulated the p-mTOR in K562R while U0126 reduced it, single IM or U0126 had no influence on p-4EBP1 in K562R, the combination of the two drugs could synergisticly reduce the p-4EBP1 and neutralize the up-regulation of p-mTOR caused by IM single agent. IM but not U0126could reduce p-cAbl and the combination of the two was more effective than IM treatment. IM alone or combination with U0126 could not regulate p-STAT5 expression in K562R. RT-PCR showed that neither IM treatment nor its combination with U0126 could change the level of lyn and erk1, 2 gene in the cell lines.

Conclusions
1. BCR-ABL-independent activation of Lyn and ERK1/2 involved in IM resistance mechanism in IM-resistant K562R cell line. Imatinib alone could up-regulated the expression of the p-Lyn, p-ERK, p-mTOR in K562R cell line. MEK1/2 inhibitor U0126 could reverse the IM resistance by reducing the expression of the p-Lyn, p-ERK, p-mTOR, p-4EBP1 of IM-resistant K562R cell line, and the combination of U0216 and Imatinib could synergisticly depress up-regulation of the p-Lyn, p-ERK, p-mTOR and p-4EBP1 caused by IM.
Grant support: National Natural Science Foundation of China (No. 30770912), Foundation of the Science & Technology Department of Sichuan Province (No. 2008SZ0017).

Disclosures:
No relevant conflicts of interest to declare.