Signal Transducer and Activator of  Transcription (STAT)5 Activation by BCR/ABL Is Dependent on Intact Src Homology (SH)3 and SH2 Domains of BCR/ABL and Is Required for Leukemogenesis

Abstract

Signal transducer and activator of transcription (STAT)5 is constitutively activated in BCR/ ABL-expressing cells, but the mechanisms and functional consequences of such activation are unknown. We show here that BCR/ABL induces phosphorylation and activation of STAT5 by a mechanism that requires the BCR/ABL Src homology (SH)2 domain and the proline-rich binding site of the SH3 domain. Upon expression in 32Dcl3 growth factor–dependent myeloid precursor cells, STAT5 activation–deficient BCR/ABL SH3+SH2 domain mutants functioned as tyrosine kinase and activated Ras, but failed to protect from apoptosis induced by withdrawal of interleukin 3 and/or serum and did not induce leukemia in severe combined immunodeficiency mice. In complementation assays, expression of a dominant-active STAT5B mutant (STAT5B-DAM), but not wild-type STAT5B (STAT5B-WT), in 32Dcl3 cells transfected with STAT5 activation–deficient BCR/ABL SH3+SH2 mutants restored protection from apoptosis, stimulated growth factor–independent cell cycle progression, and rescued the leukemogenic potential in mice. Moreover, expression of a dominant-negative STAT5B mutant (STAT5B-DNM) in 32Dcl3 cells transfected with wild-type BCR/ABL inhibited apoptosis resistance, growth factor–independent proliferation, and the leukemogenic potential of these cells. In retrovirally infected mouse bone marrow cells, expression of STAT5B-DNM inhibited BCR/ABL-dependent transformation. Moreover, STAT5B-DAM, but not STAT5B-WT, markedly enhanced the ability of STAT5 activation–defective BCR/ABL SH3+SH2 mutants to induce growth factor–independent colony formation of primary mouse bone marrow progenitor cells. However, STAT5B-DAM did not rescue the growth factor–independent colony formation of kinase-deficient K1172R BCR/ABL or the triple mutant Y177F+R522L+ Y793F BCR/ABL, both of which also fail to activate STAT5. Together, these data demonstrate that STAT5 activation by BCR/ABL is dependent on signaling from more than one domain and document the important role of STAT5-regulated pathways in BCR/ABL leukemogenesis.

STAT signaling in the pathogenesis and treatment of leukemias

“…Constitutive STAT activation has been especially well characterized in leukemias, and a growing body of evidence in both acute and chronic leukemias suggests that derangements in the STAT signaling pathway may be critical to the development of cancer in hematopoietic cells (CatlettFalcone et al, 1999;Frank et al, 1997;GouilleuxGruart et al, 1996;Nieborowska-Skorska et al, 1999;Weber-Nordt et al, 1996;Zhang et al, 1996). The multitude of hematologic cancers which demonstrate abnormal STAT activation suggests that the STAT pathway may play an important role in the pathogenesis of malignancy and may serve as an attractive target of anti-cancer therapies.…”

“…The physical interaction of Bcr/Abl and STAT5 was further delineated by Nieborowska-Skorska et al (1999) using retroviral expression of Bcr/Abl mutants in 32Dc13 cells. Speci®cally, single point and deletion mutations of the SH2 and SH3 domains were used to de®ne the necessity of these two domains for tyrosine phosphorylation and activation of STAT5 by Bcr/Abl.…”

The SOCS‐1 gene methylation in chronic myeloid leukemia patients

“…JAK/STATs are also implicated in the downstream of BCR-ABL protein, which activates the JAK/STAT pathway by its constitutive tyrosine kinase activity [3]. It has been shown that the BCR-ABL induces the STAT5 activation, which leads to leukemogenesis [4]. Loss of SOCS1 gene could play an important role in the regulation and progression of leukomogenesis in CML.…”

Progressive changes in the leukemogenic signaling in BCR/ABL-transformed cells

“…Jak-2 and IL-3/IL-5/GM-CSFRb was detected in the immunoprecipitates by Western blot assay with the speci®c antibodies (b and c, lower panels) Discussion BCR/ABL activates multiple signaling pathways leading to the transformation of hematopoietic cells (Raitano et al, 1997, Sattler andSalgia, 1997). These pathways are activated by several domains or amino acid residues of BCR/ABL such as SH1 (kinase domain), SH2, SH3, Y177, Y1294 or R1053 (Pendergast et al, 1993a,b;Afar et al, 1994;Goga et al, 1995;Cortez et al, 1995;Anderson et al, 1996;Skorski et al, 1997a;Salomoni et al, 1998;Nieborowska-Skorska et al, 1999), which are recognized as potential targets for the novel antitumor compounds. In this study we have shown that lack of activation of the BCR/ABLdependent signaling pathway(s) due to the mutation/ deletion of the particular BCR/ABL domain could be rescued by the alternative mechanisms.…”

“…This phenotype is associated with enhanced expression/activation of several e ectors (Raitano et al, 1997;Sattler and Salgia, 1997) such as Ras (Skorski et al, 1994;Sawyers et al, 1995), Rac (Skorski et al, 1998b), Raf-1 (Skorski et al, 1995a), PI-3k (Varticovski et al, 1991;Skorski et al, 1995cSkorski et al, , 1997a, Akt (Skorski et al, 1997a;Neshat et al, 2000), Bcl-2 (Sanchez-Garcia and Grutz, 1995), nuclear factor (NF)-kB (Reuther et al, 1998), CRKL (Senechal et al, 1996;Bhat et al, 1997), and STAT5 (Carlesso et al, 1996;Frank and Varticovski, 1996;Ilaria and VanEtten, 1996;Shuai et al, 1996;Chai et al, 1997;Nieborowska-Skorska et al, 1999;DeGroot et al, 1999;Sillaber et al, 2000;Horita et al, 2000). These proteins are stimulated by various functional domains/motifs of BCR/ABL (Pendergast et al, 1993a, b;Afar et al, 1994;Goga et al, 1995;Cortez et al, 1995;Anderson et al, 1996;Skorski et al, 1997a;Salomoni et al, 1998;Nieborowska-Skorska et al, 1999). Our previous studies demonstrated that signaling from SH2 or SH3+SH2 domain of BCR/ABL could be responsible for the induction of PI-3k/Akt (Skorski et al, 1997a) and STAT5 (NieborowskaSkorska et al, 1999), respectively.…”