Differentiation of imatinib -resistant chronic myeloid leukemia cells with BCR-ABL-T315I mutation induced by Jiyuan Oridonin A

Abstract

Chronic myeloid leukemia (CML) results from BCR-ABL oncogene, which blocks CML cells differentiation and protects these cells from apoptosis. T315I mutated BCR-ABL is the main cause of the resistance mediated by imatinib and second generation BCR-ABL inhibitor. CML with the T315I mutation has been considered to have poor prognosis. Here, we determined the effect of Jiyuan oridonin A (JOA), an ent-kaurene diterpenoid compound, on the differentiation blockade in imatinib-sensitive, particularly, imatinib-resistant CML cells with BCR-ABL-T315I mutation by cell proliferation assay, apoptosis analysis, cell differentiation analysis, cell cycle analysis and colony formation assay. We also investigated the possible molecular mechanism by mRNA sequencing, qRT-PCR and Western blotting. We found that JOA at lower concentration significantly inhibited the proliferation of CML cells expressing mutant BCR-ABL (T315I mutation included) and wild-type BCR-ABL, which was due to that JOA induced the cell differentiation and the cell cycle arrest at G0/G1 phase. Interestingly, JOA possessed stronger anti-leukemia activity than its analogues such as OGP46 and Oridonin, which has been investigated extensively. Mechanistically, the cell differentiation mediated by JOA may be originated from the inhibition of BCR-ABL/c-MYC signaling in CML cells expressing wild-type BCR-ABL and BCR-ABL-T315I. JOA displayed the activity of inhibiting the BCR-ABL and promoted differentiation of not only imatinib -sensitive but also imatinib -resistant cells with BCR-ABL mutation, which could become a potent lead compound to overcome the imatinib -resistant induced by inhibitors of BCR-ABL tyrosine kinase in CML therapy.

Keywords: BCR-ABL oncogene; Chronic myeloid leukemia; T315I mutation; cell differentiation; imatinib-resistant BCR-ABL mutations.

Figure 1

Anti-leukemia efficacy of JOA on BaF3-WT, BaF3-T315I, BaF3-G250E, BaF3-Y253F, BaF3-M351T, BaF3-E255K, BaF3-H296P, BaF3-M244V, BaF3-E255V, BaF3-Q252H, BaF3-F311L, BaF3-F317L, BaF3-F359V and K562cells. (A) structure of JOA, OGP46 and Oridonin. (B) The effect of JOA on the proliferation of cells treated with JOA or imatinib for 72 h. The points with error bars represent the mean ± SD. The figures are representative of three independent experiments done in triplicates.

Figure 2

JOA induced minimal signs of apoptosis in BaF3-T315I, BaF3-WT and K562 cells. (A) Analysis of apoptotic cells using the Annexin V-FITC/ PI apoptosis detection kit. (B) Bar graph showing the percentage of apoptotic cells (*P < 0.05 and **P < 0.01). Cells were incubated with 1, 2 or 4 μM of JOA or imatinib (1 or 2 μM) for 72 h.

Figure 3

JOA induced cell differentiation in BaF3-T315I, BaF3-WT and K562 cells based on cell morphology and cell membrane markers. (A) Morphological changes of cells stained with Wright-Giemsa captured by oil immersion lens (1000×). (B) The expression of cell surface antigens measured by flow cytometry. (C) Graph bars presenting the mean fluorescence intensity of antigens (**P < 0.01). Cells were incubated with 2 µM of JOA for 72 h.

Figure 4

JOA blocked cell cycle exit at G0/G1 phase in BaF3-T315I, BaF3-WT and K562 cells. (A) Cell cycle study by flow cytometry. Representative images of cells based on their profiles in G1, G0/G1, S, and G2/M phases. (B) The percentage of G0/G1 cells presented as a bar graph (*P< 0.05 and **P < 0.01). Cells were incubated with 2 μM of JOA for 24 h, 48 h or 72 h.

Figure 5

JOA significantly suppressed the colony formation of BaF3-T315I, BaF3-WT and K562 cells. (A) Colony-formation assay in methylcellulose. (B) The effect of different concentrations of JOA on colony formation.

Figure 6

Signaling pathways involved in JOA reduced cell growth in BaF3-T315I and K562 cells. (A) Volcano plots of BaF3-T315I cells and K562 cells. (B) The heatmap of all DEGs. The bars from blue to red denotes the expression levels of DEGs from low to high. (C) The heatmap of differentially expressed genes related with cell differentiation. (*P < 0.05). (D) KEGG or GSEA pathway analyses on all DEGs. BaF3-T315I and K562 cells were incubated with 2 µM of JOA for 48 h.

Figure 7

Molecular analysis of the effects of JOA on representative genes in BaF3-T315I cells and K562 cells. (A) The effect of JOA on mRNA expression of BCR-ABL, c-MYC, CCND1 or CDKN2A in K562 and BaF3-T315I cells treated with 2 μM of JOA for 24, 48 or 72 h. (B) Protein expression of BCR-ABL, p-BCR-ABL or c-MYC in both cell lines by western blotting analysis. Cells were incubated with 0.5, 1, or 2 μM of JOA or imatinib (1, or 2 μM) for 72 h. (C) Protein expression of c-MYC, CCND1 or CDKN2A in both cell lines by western blotting analysis. Cells were treated with 2 μM of JOA for 24, 48 or 72 h. (D) Graph bars showing the protein expression quantified by AI600 imager (*P < 0.05 and **P < 0.01).