The Effect Of YAP On Chronic Myeloid Leukemia Cells And Its Mechanism

Chronic myeloid leukemia(CML) is a kind of malignant clonal disorder derived from hematopoietic stem/progenitor cells. The bcr/abl fusion gene formed by the translocation of t(9; 22)(q34; q11) plays an important role in the occurrence and progression of CML. This fusion gene codes BCR/ABL oncoprotein which possesses constitutive activity of tyrosine kinase. BCR/ABL activates various downstream pathways, resulting in the malignant proliferation of myeloid cells. First been found in Drosophila, the Hippo pathway controls the organ size, balances cell proliferation and apoptosis and takes part in the regulation of cell contact inhibition by inducing cell apoptosis and inhibiting cell over-proliferation. YAP is a critical effector of Hippo pathway. As a transcription coactivator, YAP binds to transcription factors such as TEAD and Smad and regulates the transcription of c-myc and survivin. YAP has been identified to exhibit abnormal expression level and location in various cancers and is related to the malignant degree.Although studies have shown that YAP plays an important role in regulating the proliferation and apoptosis of cells, in addition its target genes c-myc and survivin are both involved in the blast crisis and prognosis of CML, its expression level and function in CML are still unclear. In the present study, we began with confirming the different expression level of YAP in cells from CML patients compared with controls and BCR/ABL+cells versus BCR/ABL-cells. Then, we explored whether YAP was regulated by BCR/ABL and we discussed the potential mechanism within. Furthermore, we detected the effects of YAP inhibition on CML cells and leukemogenesis induced by K562 and its mechanism. This study aimed at providing a new insight into the treatment of CML.The main experiments are as follows:1. In order to reveal the relationship between YAP and CML, the expression of YAP in bone marrow mononuclear cells from normal individuals( N) and CML patients in different phases( CP, AP and BP) was determined. The expression of YAP in BCR/ABL-( HL60, THP1, NB4) and BCR/ABL+(K562, KCL22, K562/G01)cells and cells transformed with( BP210, 32DP) or without( 32 D, Ba F3) p210Bcr/Abl were also detected. K562 and K562/G01 cells were treated with IM to inhibit the activity of BCR/ABL and its effects on the expression and location of YAP were evaluated. The expression of c-myc and survivin were also detected. U0126、PD98059 and LY294002 were used to find out which downstream pathway was involved in the regulation of YAP by BCR/ABL.2. si RNA and chemical inhibitor were used to suppress the function of YAP in K562 and K562/G01 cells. The effects of IM, verteporfin(VP) and the combination of IM with VP were explored in vitro. MTT assay were used to detect the proliferation of cells in different groups. Flow cytometer( FCM) was used to detect apoptosis and cell cycle distribution. Morphological features of the apoptotic cells were observed by Wright’s stain. The expression of c-Myc, survivin and proteins associate with apoptosis and cell cycle were detected by western blot.3. K562-NOD/SCID leukemic mouse model was constructed to examine the effects of IM, VP and their combination. In general, the state of the mouse, the number of white blood cell counts(WBCs), the percent of CD45+ leukemia cells in peripheral blood and the survival time were observed respectively. Mice presented obvious leukemic symptoms were executed. The weights of liver, spleen and lung were recorded. Tissues were subjected to physiological analysis after being fixed with 4% paraformaldehyde and embedded in paraffin. Infiltration of leukemic cells in these tissues was observed. The morphology of bone marrow was studied using Wright’s stain. The expression of m RNA level of c-myc and survivin were detected by RT-PCR.Results and conclusions were as follows:1. The expression level of YAP was elevated in CML patients compared with the normal controls; The protein level of YAP was much higher in BCR/ABL+(K562、KCL22、K562/G01)cells versus BCR/ABL-( HL60、THP1、NB4) ones; The ectopic expression of p210Bcr/Abl up-regulated protein level of YAP but not m RNA level; Inhibition of BCR/ABL by IM reduced expression of YAP at protein level but not at transcriptional level; IM influences the stability of YAP and promotes its degradation via ubiquitin-proteasome pathway; BCR/ABL influences the location of YAP and the inhibition of BCR/ABL suppressed the nuclear transport of YAP; IM inhibited the expression of c-myc and survivin both of which are target genes of YAP; U0126, an inhibitor of MEK-ERK signal pathway, downregulated the expression of YAP.2. si RNA was used to knockdown YAP in K562 and K562/G01 cells. The results showed that silencing of YAP significantly suppressed the proliferation of CML cells, promoted apoptosis and induced blockade of cell cycle progression from G1 to S phase. Western blot showed the expression of c-Myc and survivin, which are the target genes of Hippo pathway, and the expression of cyclin D1 were all decreased. The expression of p21, Bax, cleaved-PARP and cleaved-caspase3 were increased. Furthermore, treatment of K562 and K562/G01 cells with VP, a kind of inhibitor which can disrupt the association of TEAD-YAP complex, inhibited the proliferation, induced apoptosis and arrested the cell cycle in G1 phase. The combination of VP and IM enhanced the effects of IM on CML cells.3. The results of K562-NOD/SCID mice model showed that mice of IM, VP and the combination group displayed extended course of disease, longer survival time, slower weight losing and WBCs increasing rate. Furthermore, mice treated with IM, VP and their combination were found to have significantly reduced leukemic cell infiltration of bone marrow, liver and spleen, and lower expression level of c-myc and survivin in bone marrow. The combination of VP and IM alleviated these symptoms significantly.In conclusion, we found an elevated expression of YAP in bone marrow mononuclear cells from CML patients, BCR/ABL+( K562、KCL22、K562/G01) cells and p210Bcr/Abl transformed cell lines(BP210, 32DP) versus normal controls, BCR/ABL-( HL60、THP1、NB4), Ba F3 and 32 D, respectively. BCR/ABL regulated the expression of YAP and influenced its stability and location via MEK-ERK pathway. Genetic or pharmacological inhibition of YAP markedly inhibited the proliferation and promoted apoptosis of CML cells via decreasing the expression of proteins associated with proliferation or apoptosis such as c-Myc, survivin, cyclin D1 and increasing the expression of p21, Bax, cleaved-caspase3 and cleaved-PARP. VP synergies with IM in alleviating the symptoms of K562-NOD/SCID mice model. In this study, we evaluated the expression of YAP in CML cells and discussed the potential mechanism involved in the regulation of YAP by BCR/ABL for the first time, revealed the effects of YAP on CML cells and the related mechanism in vivo and in vitro. This study provided a new target for the treatment of CML and established a foundation for drug combinations therapy in CML.