The Gads Adaptor Protein Is Required to Mediate Lymphoid Disease Downstream of BCR-ABL,

Abstract 3750

Chronic Myeloid Leukemia is mediated by the BCR-ABL chromosomal translocation. Animal modeling experiments utilizing retroviral transduction and subsequent bone marrow transplantation have demonstrated that BCR-ABL generates a robust myeloproliferative disease that is fatal in recipient animals within 21–28 days that have received transduced marrow from donor mice primed with 5-fluorouracil. Structure/function experiments revealed that mice receiving bone marrow expressing BCR-ABL Y177F generate a T cell lymphoma with a latency of 73–116 days, demonstrating that the SH2 proteins that bind to BCR-ABL pY177 are critical for promoting myeloproliferative disease.

There are three members of the Grb2 adaptor protein family: Grb2, Gads and Grap. Biochemical experiments suggest that the Grb2 adaptor protein binds to BCR-ABL Y177 and acts to couple BCR-ABL to the Gab2 adaptor protein. In turn, Gab2 links to activation of the Ras and PI 3 kinase signal transduction pathways. Previous studies from our group revealed that Gads binds to BCR-ABL in vitro. We hypothesized that Gads plays an important role downstream of BCR-ABL. To test this objective, we performed retroviral transduction-bone marrow transplantation experiments utilizing bone marrow from wild type and Gads-deficient mice.

Thymocytes from Gads−/− mice exhibit defective proliferation in response to CD3 activation and deficits in positive and negative selection. The loss of Gads results uncoupling of Slp-76 and Lat, and impaired PLCγ phosphorylation in thymocytes. No other prominent phenotypes have been documented in this animal model.

We show that retroviral transduction-bone marrow transplantation experiments completed with 5-fluoruracil primed bone marrow display a similar latency in BCR-ABL expressing bone marrow from wild type or Gads-deficient mice.

Retroviral transduction-bone marrow transplantation experiments can also be completed with normal (unprimed) bone marrow which generates a mixed syndrome comprising myeloproliferative disease, B cell Acute Lymphoid Leukemia and macrophage tumours with a latency from 30–70 days in mice transduced with BCR-ABL. Strikingly, we observe that the lymphoid disease is absent in experiments completed with Gads−/− bone marrow expressing BCR-ABL and all mice succumb to myeloproliferative disease at 21 days. These data suggest that Gads is required for lymphoproliferative disease in this model.

We have examined signaling events in the CML-T1 and LAMA-84 CML cell lines that co-express GRB2 and GADS in order to develop models to account for our observations. GRB2 and GADS both co-immunoprecipitate BCR-ABL in both cell lines. However, there is remarkable specificity in the complexes that bind to each adaptor protein. For example, GRB2 co-immunoprecipitates the SHC adaptor protein (but not GAB2), whereas GADS co-immunoprecipitates GAB2 and SLP-76 from both CML cell lines. We are currently performing experiments to confirm these observations in BCR-ABL expressing Acute Lymphoid Leukemia patient specimens.

Our data suggests that BCR-ABL mediates Y177-dependent signaling via Grb2-dependent activation of Ras and PI 3 kinase in a myeloproliferative model of CML. However, in keeping with the important role that Gads plays in maintaining lymphocyte homeostasis, Gads mediates lymphoproliferative disease downstream of BCR-ABL, through the recruitment of specific signaling intermediates.