Friend leukemia virus provides an ideal model system for studying the multistage progression of carcinogenesis. It is composed of two viruses, the spleen focus forming virus (SFFV) and the replication competent F-MuLV. In the early phase of disease, a viral glycoprotein, gp55. encoded by SFFV interacts with and causes constitutive activation of a truncated form of the STK receptor tyrosine kinase (Sf-Stk) and the EpoR. These signals drive a polyclonal expansion of infected cells in the spleen. Mutations in p53 and integration of F-MuLV in the host genome resulting in increased transcription of the ets family members PU.1 and Fli-1, lead to leukemic transformation in the late stages of the disease. We set out to determine the mechanism by which Sf-Stk induces the early stages of transformation in this model system. To do this, we have developed an in vitro system in which primary bone marrow cells lacking Sf-Stk can be reconstituted with wild-type and mutant forms of the receptor. The cells are then infected with Friend virus and the ability of gp55 to induce cytokine-independent growth of the progenitors is assessed. Using this approach, we have shown that the kinase activity of Sf-Stk and the Grb2 binding site are critical for transformation. We have extended those studies using mice with targeted deletions in Grb2 and Gab2, that a Grb2/Gab2 complex is required downstream of Sf-Stk, leading to the recruitment and activation of Stat3. In this proposal we will identify the target cells of Friend virus in the bone marrow and spleen and investigate the role of Sf- Stk in the regulation of these cells in response to radiation or acute anemia. We propose that Friend virus co-opts normal stress response pathways to induce the rapid polyclonal expansion of infected progenitor cells. Further, we will study the role of Gab2 and Stat3 in the process of transformation of primary erythroblasts by Friend virus. Towards that end, we will utilize both genetic and biochemical approaches to map domains of Gab2 and Stat3 required for this response. We propose that this signaling pathway leads to the upregulation of PU.1 prior to retroviral insertion, resulting in the inhibition of differentiation. Taken together, these data will provide new insight into the early stages of leukemic transformation as well as the potential parallels between Friend virus-induced erythropoietic expansion and mechanisms involved in the response of these cells to stress, and provide new information regarding potential therapeutic targets.
