Mechanisms of BCR-ABL in the pathogenesis of chronic myelogenous leukaemia.

Imatinib, a potent inhibitor of the oncogenic tyrosine kinase BCR-ABL, has shown remarkable clinical activity in patients with chronic myelogenous leukaemia (CML). However, this drug does not completely eradicate BCR-ABL-expressing cells from the body, and resistance to imatinib emerges. Although BCR-ABL remains an attractive therapeutic target, it is important to identify other components involved in CML pathogenesis to overcome this resistance. What have clinical trials of imatinib and studies using mouse models for BCR-ABL leukaemogenesis taught us about the functions of BCR-ABL beyond its kinase activity, and how these functions contribute to CML pathogenesis?

Inhibition of the Raf-1 kinase by cyclic AMP agonists causes apoptosis of v-abl-transformed cells.

Here we investigate the role of the Raf-1 kinase in transformation by the v-abl oncogene. Raf-1 can activate a transforming signalling cascade comprising the consecutive activation of Mek and extracellular-signal-regulated kinases (Erks). In v-abl-transformed cells the endogenous Raf-1 protein was phosphorylated on tyrosine and displayed high constitutive kinase activity. The activities of the Erks were constitutively elevated in both v-raf- and v-abl-transformed cells. In both cell types the activities of Raf-1 and v-raf were almost completely suppressed after activation of the cyclic AMP-dependent kinase (protein kinase A [PKA]), whereas the v-abl kinase was not affected. Raf inhibition substantially diminished the activities of Erks in v-raf-transformed cells but not in v-abl-transformed cells, indicating that v-abl can activate Erks by a Raf-1-independent pathway. PKA activation induced apoptosis in v-abl-transformed cells while reverting v-raf transformation without severe cytopathic effects. Overexpression of Raf-1 in v-abl-transformed cells partially protected the cells from apoptosis induced by PKA activation. In contrast to PKA activators, a Mek inhibitor did not induce apoptosis. The diverse biological responses correlated with the status of c-myc gene expression. v-abl-transformed cells featured high constitutive levels of expression of c-myc, which were not reduced following PKA activation. Myc activation has been previously shown to be essential for transformation by oncogenic Abl proteins. Using estrogen-regulated c-myc and temperature-sensitive Raf-1 mutants, we found that Raf-1 activation could protect cells from c-myc-induced apoptosis. In conclusion, these results suggest (i) that Raf-1 participates in v-abl transformation via an Erk-independent pathway by providing a survival signal which complements c-myc in transformation, and (ii) that cAMP agonists might become useful for the treatment of malignancies where abl oncogenes are involved, such as chronic myeloid leukemias.

Characterization of Ggrb4, an adapter protein interacting with Bcr-Abl.

We report here the characterization of an adapter protein identified in a yeast 2-hybrid screen with the use of Bcr-Abl as the bait. Grb4 bound to Bcr-Abl in a variety of systems, both in vitro and in vivo, and is an excellent substrate of the Bcr-Abl tyrosine kinase. The association of Grb4 and Bcr-Abl in intact cells was mediated by an src homology (SH)2-mediated phosphotyrosine-dependent interaction as well as an SH3-mediated phosphotyrosine-independent interaction. Grb4 has 68% homology to the adapter protein Nck and has similar but distinct binding specificities in K562 lysates. Subcellular localization studies indicate that Grb4 localizes to both the nucleus and the cytoplasm. Coexpression of kinase-active Bcr-Abl with Grb4 resulted in the translocation of Grb4 from the cytoplasm and the nucleus to the cytoskeleton to colocalize with Bcr-Abl. In addition, expression of Grb4 with kinase-active Bcr-Abl resulted in a redistribution of actin-associated Bcr-Abl. Finally, coexpression of Grb4 and oncogenic v-Abl strongly inhibited v-Abl-induced AP-1 activation. Together, these data indicate that Grb4 in conjunction with Bcr-Abl may be capable of modulating the cytoskeletal structure and negatively interfering with the signaling of oncogenic Abl kinases. Grb4 may therefore play a role in the molecular pathogenesis of chronic myelogenous leukemia. (Blood. 2000;96:618-624) (Blood. 2000;96:618-624)

Functional involvement of Akt signaling downstream of Jak1 in v-Abl-induced activation of hematopoietic cells.

Activation of intracellular signaling pathways is important for cellular transformation and tumorigenesis. The nonreceptor tyrosine kinases Jak1 and Jak3, which bind to the v-Abl oncoprotein, are constitutively activated in cells transformed with the Abelson murine leukemia virus. A mutant of p160 v-Abl lacking the Jak1-binding region (v-Abl Delta858-1080) has a significant defect in Jak/STAT (signal transducers and activators of transcription) activation, cytokine-independent cell growth/survival, and tumorigenesis. To identify the pathways downstream of Jak kinases in v-Abl-mediated signaling, we examined the activation of several signaling molecules by p160 v-Abl or the v-Abl Delta858-1080 mutant. We demonstrate that, in addition to the decreased Ras activation, signaling through phosphatidylinositol-3 kinase and Akt are impaired in cells expressing mutant v-Abl. The proliferative defect of v-Abl Delta858-1080 was rescued by activated v-Akt and was also moderately rescued by activated v-H-Ras. However, constitutive active phosphatidylinositol-3 kinase (p110CAAX) did not complement this effect. Cells expressing v-Abl Delta858-1080 demonstrated reduced tumor formation in nude mice. In contrast, cells coexpressing v-Akt with v-Abl Delta858-1080 demonstrated reduced latency and increased frequency of tumor formation in nude nice compared with cells expressing v-Abl Delta858-1080 alone, whereas v-H-Ras or p110CAAX had minimum effects on tumor formation. These results suggest that Jak1-dependent Akt activation is important in v-Abl-mediated transformation.