The HSP90 inhibitor KW-2478 depletes the malignancy of BCR/ABL and overcomes the imatinib-resistance caused by BCR/ABL amplification

Abstract

Background: With the widespread clinical application of tyrosine kinase inhibitors (TKIs), an increasing number of chronic myeloid leukaemia (CML) patients have developed resistance or intolerance to TKIs. BCR/ABL is the oncoprotein of CML. HSP90 is an essential chaperone of BCR/ABL and plays an important role in protein folding and the function of BCR/ABL. Therefore, inhibiting the chaperone function of HSP90 may be an effective strategy for CML treatment and to overcome TKI resistance.

Methods: The effect of KW-2478 on CML cell viability, apoptosis and cell cycle progression was detected by CCK-8 assay or flow cytometry. The levels of BCR/ABL, HSP90 and other signalling proteins were detected by western blots. The mitochondrial membrane potential was detected by flow cytometry combined with JC-1 staining. The interaction between BCR/ABL and HSP90α was detected by coimmunoprecipitation. The effect of KW-2478 on BCR/ABL carcinogenesis in vivo was investigated in CML-like mouse models.

Results: KW-2478 inhibited growth and induced apoptosis of CML cells. KW-2478 inhibited the chaperone function of HSP90α and then weakened the BCR/ABL and MAPK signalling pathways. This treatment also caused an increase in p27 and p21 expression and a decrease in cyclin B1 expression, which led to G2/M phase arrest. The mitochondrial pathway was primarily responsible for KW-2478-induced apoptosis. KW-2478 had a synergistic effect with imatinib in growth inhibition. Notably, KW-2478 had a stronger effect on growth inhibition, apoptosis induction and cell cycle arrest of K562/G01 cells than K562 cells. KW-2478 could effectively prolong the mouse lifespan and alleviate disease symptoms in CML-like mouse models.

Conclusions: This finding demonstrated that KW-2478 had anticancer properties in imatinib-sensitive and imatinib-resistant CML cells and illustrated the possible mechanisms. This study provides an alternative choice for CML treatment, especially for TKI-resistant patients with BCR/ABL amplification and TKI-intolerant patients.

Keywords: BCR/ABL; HSP90 inhibitor; Imatinib resistance; KW-2478; Malignancy.

Fig. 1

KW-2478 inhibited the growth of BCR/ABL-positive cells with no obvious effect on BCR/ABL-negative cells. a The effects of KW-2478 on the growth of K562 and K562/G01 cells were assessed by CCK-8 assay after incubation for 24, 48 and 72 h. b The survival rate of PBMCs from two healthy individuals was detected by CCK-8 assay after incubation with KW-2478 for 48 h. c The IC50 values of KG-1a, THP1, KCL-22, K562, K562/G01, SUP-B15, 32DP-T315I and 32DP cells were detected by CCK-8 assay after 48 h of treatment with KW-2478. d, e The proliferation of CML cells after KW-2478 treatment was detected using a colony formation assay, and the results were statistically analysed. The scale bar represents 100 μm. The values are the mean standard deviation of three independent experiments. Asterisks denote statistically significant differences compared with the control group

Fig. 2

KW-2478 induced cell cycle arrest in the G2/M phase in CML cells. a After K562 and K562/G01 cells were treated with KW-2478 for 48 h, the cell cycle distribution was determined using PI staining and flow cytometry. b Following flow cytometry detection, the cell proportions in each cell cycle phase were summarized. c Levels of expression of cell cycle-related proteins, such as cyclin B1, p21 and p27, were quantitatively analysed by western blots. The results are expressed as the mean ± SD. *P < 0.05, **P < 0.01

Fig. 3

KW-2478 induced apoptosis by activating the caspase pathway in CML cells. a After K562 and K562/G01 cells were treated with KW-2478 for 48 h, flow cytometry was used to detect cell apoptosis after annexin-V-FITC/PI labelling. b The cell apoptosis rates were summarized after flow cytometry detection. c The morphology of apoptotic cells was examined after DAPI staining. The scale bar represents 10 μm. d The expression levels of cell apoptosis-related proteins, such as PARP, pro-caspase-8, cle-caspase-9 and caspase-3, were quantitatively analysed by western blots after exposure to KW-2478 for 48 h. β-Actin expression served as a loading control

Fig. 4

Apoptosis induced by KW-2478 is relevant to mitochondrial dysfunction. a The changes in mitochondrial membrane potential were detected by flow cytometry after CML cells were stained with JC-1. b The ratio of red fluorescence to green fluorescence was summarized after flow cytometry detection. c Representative images of JC-1 fluorescence imaging. The scale bar represents 50 μm. d Cytochrome C released from mitochondria was detected by western blot. e Western blotting was used to detect the expression level of the Bcl-2 protein family

Fig. 5

KW-2478 inhibited the chaperone function of HSP90α and then weakened the BCR/ABL and MAPK signalling pathways. a The expression levels of BCR/ABL, p-BCR/ABL, STAT5 and p-STAT5 in CML cells after KW-2478 treatment were detected by western blots. b The expression levels of HSP90α, HSP90β, Grp94 and Hsp70 in CML cells after KW-2478 treatment were quantitatively analysed by western blot. c The mRNA level of BCR/ABL was detected with qRT-PCR. β-Actin was used as an internal control. d The mRNA level of HSP90α was detected with qRT-PCR. e The interaction between HSP90α and BCR/ABL was analysed by Co-IP assays with no KW-2478 treatment. f The influence of KW-2478 on the interaction between HSP90α and BCR/ABL was analysed by Co-IP assays. g Molecules in the MAPK signalling pathway, such as Ras, phospho-Raf1, Mek1/2, phosphorylated and total Erk1/2, were analysed by western blots. The results are expressed as the mean ± SD. *p < 0.05

Fig. 6

KW-2478 synergistically functioned with imatinib to induce apoptosis and growth inhibition in CML cells. a A CCK-8 assay was used to determine the combined effects of KW-2478 and imatinib on cell viability of K562 and K562/G01 cells. b The combination index of K562 or K562/G01 cells was analysed using CompuSyn software. c Following treatment with KW-2478, imatinib or both, cell apoptosis was detected by flow cytometry. d Statistical analysis of the apoptosis rates of K562 and K562/G01 cells treated with KW-2478, imatinib or both

Fig. 7

KW-2478 had effective antitumor activity in imatinib-sensitive and imatinib-resistant CML-like mouse models. a The maximum WBC counts of mice were recorded. b, c The liver and spleen weights of mice were measured. The results are expressed as the mean ± SD. *p < 0.05, **p < 0.01. d Images of the liver and spleen are displayed. e Murine liver and spleen infiltration was analysed by HE staining. The scale bar represents 50 μm. f Cells from mouse bone marrow, liver, and spleen were stained by Wright’s stain and then photographed under a microscope. The scale bar represents 20 μm. g The expression of BCR/ABL in each tissue was detected by immunofluorescence assays. The scale bar represents 10 μm. h The expression levels of BCR/ABL and HSP90 family proteins from mouse bone marrow cells were detected by western blots. i The survival curves of mice were analysed by the Kaplan–Meier method

Fig. 8

Schematic diagram of the function and mechanisms of KW-2478 in CML cells. KW-2478 degrades BCR/ABL and downstream signalling proteins by inhibiting the binding of HSP90α and BCR/ABL. This molecule inhibits the growth of CML cells by inhibiting the MAPK signalling pathway. KW-2478 causes cytochrome C release and mitochondrial dysfunction to activate the caspase pathway and then induces apoptosis in CML cells