Inhibition of YAP suppresses CML cell proliferation and enhances efficacy of imatinib in vitro and in vivo

Abstract

Background: Yes-associated protein (YAP), an essential component of Hippo pathway, was identified as an oncoprotein which participated in the progression of various malignancies. However, its role in chronic myeloid leukemia (CML) remains to be further clarified.

Methods: The expression of YAP in CML cells was determined by western blotting. Next, the effects of YAP knockdown and YAP inhibitor on CML cells were evaluated by MTT assay, flow cytometry (FCM) and Wright's staining. Moreover, K562 induced mice model was employed to further investigate the role of YAP in vivo.

Results: YAP was overexpressed in CML cells. Knockdown of YAP by si-RNA or inhibition the function of YAP using verteporfin (VP) not only inhibited the proliferation, induced the apoptosis of CML cells but also reduced the expression of YAP target genes c-myc and survivin. Additionally, VP enhanced the efficacy of imatinib (IM) in vitro and suppressed leukemogenesis in vivo.

Conclusion: Our results indicate that YAP may play an important role in the proliferation and leukemogenesis of CML cells. Genetic or pharmacological inhibition of YAP provides a novel treatment strategy for CML.

Keywords: Bcr/Abl; Chronic myeloid leukemia; IM; Leukemogenesis; Verteporfin; YAP.

Fig. 1

Expressions of YAP in BMMNCs from CML patients and different leukemic cell lines. a The protein expression of YAP was up-regulated in BMMNCs separated from CML patients in different phases (n = 9) compared with that from the healthy individuals (n = 5). b the mRNA level of YAP showed no significant difference between CML patients and the healthy individuals. c YAP was up-regulated in BCR/ABL positive cell lines K562, KCL22 and K562G01compared with BCR/ABL negative cell lines HL60, NB4 and THP. d The ectopic expression of BCR/ABL dramatically up-regulated YAP at protein level (**P < 0.01) but not mRNA level (e). Inhibition of BCR/ABL by IM down-regulated YAP at protein level (f) (**P < 0.01) but not mRNA level (g)

Fig. 2

Silencing of YAP inhibits the proliferation of CML cells. a Knockdown efficiency of siRNA was validated by western blotting analysis. b Cell viability was determined by MTT, viability of siRNA transfected K562 and K562/G01 decreased in a time dependent manner (**p < 0.01). c Cell cycle was analyzed by flow cytometry at 48 h after transfection. d The expression of P21 and Cyclin D1 were analyzed by western blot

Fig. 3

Silencing of YAP induces apoptosis of CML cells. a The percentage of apoptotic cells were examined by FCM. b Morphological features of the cell apoptosis induced by si-YAP (50 nM). c The expression of Bax, cleaved caspase-3 and PARP were analyzed by western blot. Knockdown of YAP by siRNA down-regulated c-Myc and survivin both at protein (d) and mRNA levels (e) (**P < 0.01)

Fig. 4

YAP inhibitor enhances the effect of IM on CML cells. a K562 and K562/G01 cells were treated with different dose of VP and the inhibitory rate was increased as a dose-dependent manner. b K562 and K562/G01 cells were treated with VP (10 μM) with or without 2 μM IM for 24 h, and cell viability was evaluated by MTT assay. c Cell cycle distribution was examined by FCM. d Apoptotic rate was analyzed by FCM. e After K562 and K562/G01cells were treated with VP, IM or their combination for 24 h, the expression of c-Myc, survivin, P21, Cyclin D1, Bax and the Cleaved caspase-3 and PARP were detected by western blot. *p < 0.05 and **p < 0.01 versus control group, #p < 0.05 versus IM alone

Fig. 5

YAP inhibitor potentiates the efficacy of IM on leukemogenesis in vivo. a Total WBC count in PBS, VP, VP and IM group were determined. b, c Mean liver and spleen weight of mice in different group were quantified. d Representative images of livers and spleens from four groups. e Histologic sections of liver and spleen were stained with H&E and bone marrow cells from mice in each group were stained with Wright’s staining. f Percentage of Human CD45⁺ cells in murine bone marrow were detected by flow cytometry. g Survival curves were analyzed by Kaplan–Meier methods. *p < 0.05 and **p < 0.01 versus control group, #p < 0.05 and ##p < 0.01 versus IM alone