BCR-ABL and Src Family Kinases Induce YAP Tyrosine Phosphorylation Resulting in Survivin and Cyclin D1 Expression in Chronic Myeloid Leukemia Cells

Chronic myeloid leukemia (CML) is caused by the BCR-ABL fusion protein. To date, several downstream signaling pathways of BCR-ABL have been reported to underlie the leukemogenesis of CML such as the JAK/STAT pathway, the PI3K/AKT pathway, and the Grb2/MAPK pathway. Furthermore, the Src family kinases (SFKs), especially Hck, Lyn and Fyn, have been suggested to be involved in BCR-ABL-induced transformation. Although these studies have revealed important aspects of the downstream signals of BCR-ABL, the detailed molecular mechanism of CML has not been thoroughly elucidated.

Yes-associated protein (YAP) is a transcriptional cofactor that functions as an effector of the Hippo pathway which regulates cell growth and survival. In the classical Hippo pathway, YAP phosphorylated at serine 127 (S127) by LATS1/2 is bound to 14-3-3 and prevented from nuclear translocation. Apart from this serine/threonine phosphorylation, YAP undergoes phosphorylation at several tyrosine residues by various kinases to be activated. SFKs can phosphorylate and activate YAP, which has been demonstrated in some tumors. Among the several possible phosphorylated tyrosine residues, the phosphorylation at Y357 (p-Y357) has been demonstrated to be the most important for tumorigenesis. Therefore, it is possible that BCR-ABL directly or indirectly phosphorylates YAP through SFKs and thus activated YAP is translocated into the nucleus and together with TEAD induces expression of genes necessary for cell growth and survival.

In the present study, we investigated the effects of imatinib and an SFK-specific inhibitor RK-20449 on viable cell number, YAP p-Y357 and expression of Survivin as well as Cyclin D1 in CML-derived cell lines in comparison with AML-derived cell lines. Furthermore, we established BCR-ABL stable transfectants and the control lines derived from TF-1, a factor dependent human erythroleukemia cell line, in order to verify our results obtained with CML-derived cell lines. We first checked the phosphorylation status of YAP and found that it was constitutively phosphorylated at tyrosine 357in CML-derived cell lines (TCC-S and K562) but not in AML-derived cell lines (HL-60 and KG-1a). Treatment with imatinib or RK-20449 inhibited cell growth and decreased tyrosine phosphorylation of YAP in both CML lines. Expression of Survivin or Cyclin D1 was decreased at least in TCC-S but not in AML cell lines. Furthermore, we established BCR-ABL stable transfectant and control empty vector transfectant from TF-1, a factor dependent human erythroleukemia cell line, in order to verify our results obtained with CML cell lines. YAP was phosphorylated at Y357 constitutively in BCR-ABL stable transfectant but not in control transfectant, and treatment with imatinib or RK-20449 inhibited cell growth, YAP tyrosine phosphorylation, and expression of Cyclin D1 in BCR-ABL stable transfectant.

These results suggest that BCR-ABL induces tyrosine phosphorylation of YAP presumably through Src family kinases, which results in expression of Survivin and Cyclin D leading to leukemogenesis in CML cells. We have not determined which SFK is involved in the downstream signaling of BCR-ABL. Overexpression experiments in HEK293T have indicated that Lck, Lyn and Fyn can phosphorylate YAP at Y357 without BCR-ABL. However, it remains to be determined which SFK is activated by BCR-ABL and practically involved in YAP phosphorylation resulting in leukemogenesis. Further studies are required to specify the relevant SFKs and disclose the downstream signaling from activated YAP.