Friend spleen focus-forming virus (SFFV) causes a rapid, multi-stage erythroleukemia in mice due to expression of its unique envelope glycoprotein. It provides an important model for understanding the molecular changes that result in the hyperplastic, blast crisis and metastatic phases of leukemia and for testing therapeutic strategies to block each stage. Our previous studies demonstrated that during the first stage of SFFV-induced disease, the viral envelope protein, SFFV gp55, forms a complex with the erythropoietin receptor (EpoR) and a short form of the receptor tyrosine kinase Stk (sf-Stk). This complex causes constitutive activation of various components of the Epo signal transduction pathway and Epo-independent erythroid cell proliferation, survival and differentiation. Recently, we discovered that co-expression of SFFV gp55 and sf-Stk is sufficient to transform NIH 3T3 and primary fibroblasts. Our subsequent studies indicate that sf-Stk expression is required for maintenance of the transformed phenotype of SFFV gp55-expressing fibroblasts, and that PU.1, which is essential for transformation of erythroid cells by SFFV, plays no role in transformation of fibroblasts by SFFV gp55/sf-Stk. We also took advantage of the SFFV gp55/sf-Stk-transformed fibroblasts to examine the activation of signal transduction pathways by SFFV gp55 in the absence of the EpoR. Like SFFV-infected erythroid cells, SFFV gp55/sf-Stk- transformed fibroblasts express high levels of phosphorylated MEK, ERK, PI 3-kinase, Gab1/2, Akt, JNK and STAT3, but unlike virus-infected erythroid cells they fail to express phosphorylated STATs 1 and 5, which may require involvement of the EpoR. In addition, the p38 MAPK stress response is suppressed in the transformed fibroblasts. We further showed that the signal transducing molecules activated in fibroblasts by SFFV-activated sf-Stk can be targeted with small molecule inhibitors to modulate proliferation and/or transformation, and that it may be possible to target sf-Stk directly with the flavonoid luteolin. Our results indicate that sf-Stk is a molecular effector of transformation that could be targeted directly or with agents against its downstream signal transducers. We are now extending our studies to determine if the human counterpart of sf-Stk, sf-RON, is activated in any human malignancies. Our preliminary data indicates that sf-RON is expressed in certain types of human cancer cell lines and studies are in progress to determine if blocking the kinase in these cells will alter their transformed phenotype. In addition to studying the effects of SFFV gp55 on erythroid cell proliferation and differentiation, we are also studying the second stage of SFFV-induced leukemia for molecular changes associated with transformation and metastasis. Transformation of erythroid cells by SFFV is associated with activation of the transcription factor PU.1 by retroviral integration, and our recent studies suggest that this results in high expression of the hematopoietic phosphatase SHP-1. This subsequently leads to a specific block in the phosphorylation of STAT1, a transcription factor important for erythroid cell differentiation that is activated by SFFV in the first stage of the disease. We recently demonstrated that SFFV-transformed erythroleukemia cells can also undergo further molecular changes which allow them to metastasize to the bone marrow. Due to their failure to differentiate, they proliferate to such high levels in the bones of the skull and vertebrae that they breech the bone and enter into the meninges of the brain and spinal cord, causing meningeal leukemia associated with hind limb paralysis. Since meningeal leukemia is a common complication of human leukemia, we are now developing this as an animal model to study the mechanisms by which leukemic cells metastasize to the central nervous system and to test therapies to block these events. Our recent studies have focused on identifying differences in the leukemic cells from their non-transformed counterparts that may be associated with their ability to cause meningeal leukemia. The SFFV-transformed erythroleukemia cells were shown to secrete high levels of vascular endothelial growth factor (VEGF) and to preferentially adhere in vitro to fibronectin. Gene prolifing showed changes in expression of genes encoding angiogenic growth factors, oncoproteins and adhesion molecules as well as genes involved in the synthesis or degradation of extracellular matrix components. When bone sections of diseased mice were examined, excessive and pathological angiogenesis could be seen in the marrow compared with controls. Studies are in progress to test anti-angiogenesis drugs as well as other pharmacological agents for their ability to block metastasis of the SFFV transformed erythroleukemia cells to the bone marrow and the development of meningeal leukemia.
PVC-211 murine leukemia virus (MuLV), a variant of an erythroleukemia-inducing retrovirus, causes a rapid neurodegenerative disease when injected into newborn rats. This is an important model for understanding how retroviruses can undergo genetic changes that alter their interaction with cells in the host to cause novel biological effects. Our previous studies demonstrated that the envelope protein of PVC-211 MuLV had undergone subtle changes in its receptor binding domain that allow it to enter the central nervous system by efficiently infecting brain capillary endothelial cells (BCEC). These changes created a unique heparin-binding domain in the viral envelope protein that may allow the virus to bind strongly to the surface of BCEC via heparin-like molecules, increasing the probability that the virus will bind to its cell surface receptor on these cells and efficiently infect them. Our more recent studies have concentrated on understanding the molecular events that occur after PVC-211 MuLV infects BCEC. We previously showed that virus-infected BCEC express high levels of inducible nitric oxide synthase and the chemokine LIX and show evidence of nitric oxide production. Our recent studies demonstrate that early in the course of the disease, before neuronal damage has occurred, the number of blood vessels increases in the cerebellum and brain stem, the sites of later spongiform neurodegeneration; the vessels in the brain become leaky; and the cerebellum expresses high levels of vascular endothelial growth factor (VEGF), suggesting brain hypoxia is occurring. At the same time, microglia in the virus-infected brain become activated and the brain and blood express elevated levels of MIP-1 alpha, a chemokine known to be secreted from activated microglia. Once the pathogenic changes can be detected in the cerebellum of virus-infected rats, one can detect elevated levels of tissue plasminogen activator (tPA), which could be responsible for neuronal death. We are currently using NMR spectroscopy to determine if elevated levels of any potentially neurotoxic brain metabolites can be detected in virus-infected brains compared with control brains. Studies are in progress to evaluate several therapeutic strategies to prevent or delay virus-induced neurodegeneration, including (1) treatment with erythropoietin, which may suppress the effects of early hypoxia, (2) treatment with an inhibitor of iNOS to block nitric oxide production, (3) treatment to block VEGF to avoid blood vessel damage and (4) treatment to deplete or block the activation of microglia, to eliminate any neurotoxic factors that they may secrete
