Role of the STK Receptor in Erythropoiesis

描述（申请人提供）：Friend白血病病毒为研究癌变的多阶段进展提供了理想的模型系统。它由脾灶形成病毒（SFFV）和复制能力强的F-MuLV两种病毒组成。在疾病的早期阶段，一种病毒糖蛋白gp55。由SFFV编码的蛋白与STK受体酪氨酸激酶（Sf-Stk）和EpoR的截断形式相互作用并引起组成性激活。这些信号驱动脾脏中感染细胞的多克隆扩增。p53的突变和宿主基因组中F-MuLV的整合导致ets家族成员PU.1和fl -1的转录增加，导致疾病晚期的白血病转化。我们着手确定Sf-Stk在该模型系统中诱导早期转变的机制。为了做到这一点，我们开发了一个体外系统，在这个系统中，缺乏Sf-Stk的原代骨髓细胞可以用野生型和突变形式的受体重建。然后用Friend病毒感染细胞，评估gp55诱导祖细胞不依赖细胞因子生长的能力。使用这种方法，我们已经证明了Sf-Stk和Grb2结合位点的激酶活性对转化至关重要。我们对Grb2和Gab2靶向缺失的小鼠进行了扩展研究，发现Sf-Stk下游需要Grb2/Gab2复合物，从而导致Stat3的募集和激活。在本研究中，我们将在骨髓和脾脏中确定Friend病毒的靶细胞，并研究Sf- Stk在这些细胞对辐射或急性贫血的反应中的调节作用。我们认为Friend病毒通过正常应激反应途径诱导受感染祖细胞快速多克隆扩增。进一步，我们将研究Gab2和Stat3在Friend病毒转化原代红母细胞过程中的作用。为此，我们将利用遗传和生化方法来绘制这种反应所需的Gab2和Stat3结构域。我们认为这一信号通路在逆转录病毒插入之前导致PU.1的上调，从而抑制分化。综上所述，这些数据将为白血病转化的早期阶段以及Friend病毒诱导的红细胞增生与这些细胞对应激反应的机制之间的潜在相似之处提供新的见解，并为潜在的治疗靶点提供新的信息。

DESCRIPTION (provided by applicant): 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.