BCR-ABL1 mediates up-regulation of Fyn in chronic myelogenous leukemia

Abstract

Chronic myelogenous leukemia (CML) in IntroductionThe management of chronic myelogenous leukemia (CML) has been revolutionized by kinase inhibitors that were developed in response to cues from biologic studies of the BCR-ABL1 oncogene. However two challenging problems persist: the progression of the disease to blast crisis and resistance to kinase inhibition. 1 Continued investigation of BCR-ABL1 kinase signaling will provide insight into these problems. Members of the Src kinase family, which regulate proliferation, differentiation, and motility, 2 are known downstream targets of BCR-ABL1. In myeloid cell lines, BCR-ABL1 activates Lyn and Hck. 3,4 Several reports link growth, survival, and imatinib resistance of Philadelphia chromosome-positive (Ph ϩ ) leukemias to Lyn kinase expression and activation. 5,6 However, reports examining Fyn, a ubiquitously expressed Src family member, are sparse. Of note, phase-specific gene expression in CML using microarray analyses revealed that Fyn gene expression was linked to imatinib relapse. 7 In addition, a separate study using combined systems biology and gene expression approaches in Ph ϩ acute lymphoblastic leukemia (ALL) specimens identified Fyn as a hub for signaling. 8 Here we show that Fyn protein expression is increased in patients with blast-crisis CML compared with chronic-phase disease. By examining effects of silencing Fyn using shRNA, we find that Fyn transduces a mitogenic signal. Collectively, our results identify a novel effect of BCR-ABL1-up-regulation of Fyn-and delineate consequences of the observed up-regulation. MethodsPatient specimens were used for this study and were collected after informed consent was obtained in accordance with the Declaration of Antibodies, chemicals, and cell linesAntibodies were purchased from sources outlined in Document S1 (available on the Blood website; see the Supplemental Materials link at the top of the online article). Imatinib was kindly provided by Dr Elisabeth Buchdunger at Novartis Pharmaceuticals (Basel, Switzerland). Murine growth factor-dependent pro-B lymphoid BaF3 cell lines transformed with vector, wild-type BCR-ABL1, or imatinib-resistant mutant BCR-ABL1 were kindly provided by Dr Charles Sawyers 9 and were cultured as previously described. 9 K562 cells, TonB210 cells stably expressing a tetracyclineinducible BCR-ABL1 expression vector (kindly provided by Dr George Daley, Children's Hospital Boston, Harvard Medical School, MA), 10 and mouse 32D and 32Dp210 cells were maintained in RPMI1640 medium with 10% FBS supplemented. Mouse 32D cells were supplemented with 10% WEHI-cultured conditioned medium as a source of interleukin-3 (IL-3) in addition to 10% FBS. Design of shRNA to FynK562 cells were transfected with Fyn shRNA and control vectors (TranSilent human shRNA from Panomics, Redwood, CA) using the Nucleofector system kit V and transfection program T-16 (Amaxa Biosystems, Cologne, Germany). Lentiviral knockdown of Fyn and rescue design is detailed in Document S1. To generate the rescue construct, 4 nucleo...

Gene expression profiling of imatinib and PD166326-resistant CML cell lines identifies Fyn as a gene associated with resistance to BCR-ABL inhibitors

“…We established here that Fyn is up-regulated in imatinib-and PD166326-resistant K562 cell lines, linking Fyn overexpression to acquisition of resistance to BCR-ABL inhibitors. This is coherent with the fact that resistance to imatinib is a common feature in patients with accelerated phase or blast crisis (12) and with its potential role in transformation (37). The IM-R K562 cells described by Donato et al retained active BCR-ABL kinase activity that was inhibitable by imatinib (21), suggesting that BCR-ABL was not coupled to proliferation or survival of K562-R cells.…”

“…Taken together, our findings are in favor of an important role of Fyn in the resistance to BCR-ABL inhibitors. Ban et al also reported that most of the CML cells from patients in blast crisis were positive for Fyn, whereas the majority of chronic and accelerated specimens were negative (37). Of note, BaF3 cells expressing imatinib point mutants of BCR-ABL retained the ability to up-regulate Fyn (37).…”

“…Indeed, if the contribution of the Src kinases Lyn and Hck in BCR-ABL-mediated leukemogenesis and resistance to BCR-ABL inhibitors is well established, little is known about the role of Fyn in this context. Recently Ban et al (37) reported, using tissue arrays, that Fyn expression is increased in CML blast crisis as compared with chronic-phase patients. Moreover, BCR-ABL overexpression results in an up-regulation of Fyn protein and mRNA.…”

“…Recently Ban et al reported that Fyn was significantly increased in CML blast crisis as compared with chronic phase and that BCR-ABL overexpression results in up-regulation of Fyn protein and mRNA (37). We thus investigated the 6 /mL) were incubated for 30 min at 37°C with 1 μmol/L imatinib or 3 nmol/L PD166326.…”

microRNA-125a-3p reduces cells proliferation and migration by targeting Fyn

“…Fyn inhibition by siRNA induces cell cycle arrest and impairs cell viability and migratory ability Because Fyn is considered an important mediator of mitogenic signaling and a regulator of cell cycle, growth and proliferation (Cowden Dahl et al, 2009;Ban et al, 2008;Posadas et al, 2009), we conducted experiments where Fyn expression was inhibited by siRNA and assessed the effect on proliferation, viability and migration of the cells. Indeed, by using Fyn-siRNA (si-Fyn) we were able to obtain an 80% decrease in the expression of Fyn mRNA (Fig.…”