Samples were lysed on ice for 1 h using Triton-X-100 lysis buffer as described above. short latency myeloid disease within 3C4 weeks of transplant, while wild-type mice succumb to both a longer latency lymphoid and myeloid diseases. We report that GADS mediates a unique BCR-ABL complex with SLP-76 in BCR-ABL-positive cell lines and B-ALL patient samples. These data suggest that GADS mediates lymphoid disease downstream of BCR-ABL through the recruitment of specific signaling intermediates. gene from chromosome 9,3,4 forming cGAMP the fusion protein BCR-ABL, resulting in constitutive activation of the ABL tyrosine kinase.5 BCR-ABL is the causative agent in Ph chromosome-positive (Ph+) leukemias, including chronic myeloid leukemia (CML)3,4 and B-cell acute lymphoblastic leukemia (B-ALL).6 The pathogenesis of CML caused by BCR-ABL can be modeled in the murine bone marrow transplant (BMT) assay. BCR-ABL-transduced bone marrow progenitors (from 5-fluorouracil (5-FU)-primed donors) injected into lethally irradiated recipient mice gives rise to fatal myeloproliferative disease (MPD) that resembles human CML within 3C4 weeks.7,8 When whole bone marrow from non-5-FU-primed donors is used for transduction and transplantation, a model of BCR-ABL-mediated B-ALL is observed. In the latter model, BMT recipients succumb to a mixture of disease phenotypes, including a B-cell disease that resembles human B-ALL.9 Tyrosine (Y) 177 of BCR-ABL is critical to the development of CML-like disease in mice.10C12 Phosphorylation of this residue is responsible for the SH2 domain-dependent binding of the adapter protein Grb2 that serves to couple BCR-ABL to the Gab2 adapter protein.13 In turn, Gab2 recruitment leads to the activation of the Ras14,15 and phosphoinositide 3 kinase16 signaling pathways required for BCR-ABL-mediated transformation.3,4,14,17 Gads (Grb2-related cGAMP adapter protein downstream of Shc) is a Grb2 family member and has been shown to interact with BCR-ABL through studies.18,19 Like its family members Grb2 and Grap,20 Gads possess a central SH2 domain, flanked by two SH3 domains.21 Although the SH2 domains of all three family members have cGAMP similar binding specificities,11,18,22 the carboxy terminal SH3 domain of Gads possesses unique binding specificity that allows for interaction with the adapter protein SLP-76 (Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa).23 Both Gads24 and SLP-7625 are required for normal T-cell development. Upon T-cell receptor (TCR) activation, the Gads-SLP-76 complex is recruited to tyrosine-phosphorylated LAT (linker for activation of T cells) via the SH2 domain of Gads.22,26 The formation of this adapter protein complex allows for FLJ31945 the aggregation of signaling complexes critical to T-cell activation, including those leading to cytoskeletal changes, interleukin (IL)-2 gene expression and proliferation.27 Previous studies have sought to clarify the molecular events that differentiate Ph + myeloid disease (CML) and lymphoid disease (B-ALL), which have led to the identification of unique activation of SRC family tyrosine kinases in lymphoid disease.28 Compared with CML, Ph + B-ALL has historically been resistant to therapy and associated with poor clinical outcomes.29C32 The identification of unique pathways that distinguish the two diseases could provide insight into the identification of novel therapeutic targets to treat Ph + ALL. Due to its essential role in lymphocyte signaling and development and its ability to interact with BCR-ABL through its SH2 domain, we tested whether Gads could serve as a candidate for mediating BCR-ABL-mediated lymphoid disease. Through the use of the murine BMT assay, we determined that Gads is required cGAMP for BCR-ABL-mediated lymphoid disease but is inconsequential for BCR-ABL-mediated myeloid disease. GADS is expressed in a subset of CML cell lines and B-ALL patient samples and we show in these samples that GADS associates with both BCR-ABL and SLP-76. Together these data provide evidence that signaling through Gads is critical to the development of BCR-ABL-mediated lymphoid disease. MATERIALS AND METHODS DNA constructs The Mig210 construct was used to express the p210 isoform of BCR-ABL in BMT experiments. The vesicular stomatitis virus G and pSV?env ? plasmids were used to generate high titer retroviral supernatants. Animals Studies were approved by the Animal Care Committee at the cGAMP Ontario Cancer Institute (OCI) and The Center for Phenogenomics, Toronto. ON, Canada. The generation of Gads(?/?) mice was previously described.24 Gads (?/?) mice were back-crossed to the BALB/c for eight generations. Wild-type BALB/c donor and recipient mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). All donor and recipient mice were used at 6C8 weeks of age. Generation of retroviral stocks Calcium phosphate transfections (CalPhos Mammalian Transfection Kit, Clontech, Mountain View, CA, USA) of 293T cells were performed with retrovirus, pSV?env? packaging.