Clonal diseases of large granular lymphocytes (LGL) are characterized by lymphocytosis and autoimmune manifestations including rheumatoid arthritis, pulmonary hypertension, and bone marrow failure. Several related syndromes have similar pathology involving the bone marrow including aplastic anemia (AA), paroxysmal nocturnal hemaglobinuria (PNH), and a subset of patients with Myelodysplastic Syndrome (MDS). Clinical improvement occurs after immunosuppressive therapy (IST) suggesting that activated T-cell induce this syndrome. The broad long-term objectives of this proposal are to improve the diagnosis and treatment of patients with LGL leukemia and other diseases associated with hyperactive memory T-cells. In our earlier studies of survival regulation in this disease, our lab was the first to demonstrate constitutive activation of signal transducer and activator of transcription 3 (STAT3) transcription factor (STAT3) DNA binding. STAT-family proteins, including STAT3, play a critical role in many biological processes. They have proven oncogenic potential in several forms of cancer including breast, prostate, pancreas, leukemia and lymphoma. Until recently, the mechanism causing STAT3 activation was unknown. Recurrent mutations in the STAT3 Src homology-2 (SH2) domain were identified through exome sequencing by two independent international groups in 2012. More than 80% of the STAT3 mutations result in amino acid substitutions at position Y640F or D661Y (called mutSTAT3 throughout the application). Although these two amino acids have no known function in STAT3 activation, they have the potential to alter major protein-protein interactions with activated cytokine receptors or to strengthen carboxy-terminal phosphotyrosine 705 (Y705)-mediated dimerization. There is a known linkage between STAT3 and memory T-cell differentiation. A dominant negative STAT3 mutation occurs in patients with Hyper IgE Syndrome (HIES, or Job Syndrome). In these patients, a reduction in central memory T-cell development suggests that STAT3 may be involved in the regulation of memory T-cell differentiation. Mice with targeted deletion of STAT3 in T-cells also have a loss of central memory T-cells and an accumulation of inactive terminal memory cells during viral infection. Overexpression of active STAT3 in T-cells will close an important knowledge gap in the precise role of STAT3 in memory T-cell differentiation. Since the Y640F and D661Y represent the first naturally-occurring mutations with potential for gain-of-function, we will be able to address important questions with human disease relevance. The hypothesis to be tested in this application is that the naturally-occurring STAT3 mutations (Y640F and D661Y) alter SH2 domain structural properties leading to constitutive activation. Expression of this activated form of STAT3 then contributes to aberrant survival and expansion of T-cells with an effector memory phenotype capable of injuring normal tissues. Our research design will include three specific aims (SA): 1) to prove that naturally-occurring STAT3 mutations (mutSTAT3) increase survival by altering the STAT3 activation threshold, 2) to prove that Y640F and D661Y alter SH2 peptide domain interactions and serve as a unique target for drug design and, 3) to confirm our hypothesis that mutSTAT3 induces T-cell activation in memory T-cells to induce survival and tissue damage. Due to the prevalence and health care costs associated with autoimmune cytopenias, and the overwhelming devastation and poor treatment options of autoimmune diseases in Veterans, we believe our project is broadly relevant to the Veteran population and is focused on a critical health problem of interest to VA R&D.
Large granular lymphocyte (LGL) leukemia occurs primarily in older individuals and is associated with poor blood formation. Blood counts have been shown to recover in some patients after treatments that block a hyper-active immune response. A better basic understanding of the abnormalities in the immune system is needed to understand why this form of treatment works and to develop more effective therapies. In our study, we show genetic and functional problems in the immune system that support a novel understanding of the disease. We will expand on our early findings and perform experiments in the laboratory to determine exactly how this genetic abnormality cause the disease features. We will screen drugs that block the genetic abnormality and determine if this reverses pathology. These studies have the potential to improve the health care of veterans with LGL leukemia and millions of patients with related bone marrow diseases.
|Mailloux, Adam W; Zhang, Ling; Moscinski, Lynn et al. (2013) Fibrosis and subsequent cytopenias are associated with basic fibroblast growth factor-deficient pluripotent mesenchymal stromal cells in large granular lymphocyte leukemia. J Immunol 191:3578-93|