Anemia affects ~1.6 billion people worldwide, imposing an enormous burden on medical resources. Bone marrow failure (BMF) leading to severe anemia occurs in a large number of inherited and acquired hematologic disorders. Despite progress in identifying the molecular mechanisms underlying inherited BMF syndromes, the genetic defect remains unexplained in many cases. Scat (severe combined anemia and thrombocytopenia) is a unique mouse model characterized by pancytopenia and BMF. Significantly, the scat phenotype is episodic. Homozygotes are severely affected at birth, but a remarkable, spontaneous remission ensues, wherein the disease phenotype reverts to normal. Notably, BMF in humans is known to remit spontaneously, as well. In scat a second ?crisis? invariably follows, and 90% of the mice die by P36 due to catastrophic hematopoietic failure in the bone marrow and spleen. We showed that scat carries a mutation in a gene not previously recognized as critical to vertebrate erythropoiesis, Rasa3. Moreover, we recapitulated the dramatic scat phenotype in zebrafish using rasa3-specific morpholinos and in cultured human CD34+ cells transfected with RASA3-specific siRNAs, confirming a critical, conserved and non-redundant role for RASA3 in blood formation. We hypothesize that a feedback regulatory mechanism(s) critical to blood formation is defective in scat. Here, we propose to utilize detailed erythroid-focused approaches integrated with broad systems level approaches to identify the mechanisms and functional gene networks that mediate the changing scat phenotype. Specifically, we will: (1) Determine how RASA3 loss of function alters erythroid cell properties (e.g., oxidative status, cell cycle status, apoptosis, mitophagy, activation status of signaling effectors) in scat mice in crisis compared to scat remission and wild type (WT) during terminal differentiation; (2) Establish the role of RASA3 in the physiology and pathophysiology of human erythropoiesis utilizing stable lentiviral knock-downs in CD34+ cells and screening for RASA3 mutations in DNA of de-identified patients with BMF of unknown origin; (3) Obtain expression (RNAseq, miRseq) and phospho(p)-proteome profiles to identify gene expression and post-transcriptional differences during disease progression using tissues and purified cell populations from distinct scat disease stages and from WT controls and (4) Analyze and integrate all data to identify and prioritize candidate genes; initiate functional characterization of the most compelling candidate genes. Identifying mechanisms contributing to severe hematopoietic crisis in scat offers an entry point into gene networks relevant to BMF. Identifying mechanisms underlying the dramatic re-initiation of hematopoiesis, i.e., the complete reversal of hematopoietic failure that occurs during remission will provide new therapeutic targets for acquired and inherited BMF syndromes that will likely benefit other types of anemia, as well.
Anemia affects approximately 1.6 billion people worldwide, imposing an enormous burden on medical resources. Of the inherited anemias, the bone marrow failure (BMF) syndromes represent a particularly severe class with high morbidity and mortality. Many patients are non-responsive to treatment and become transfusion dependent. Notably, anemias share common etiologies in mice and man. Thus, mice are excellent model organisms in which to study mechanisms of anemia and potential therapies. The scat (severe combined anemia and thrombocytopenia) mouse model is a unique model of BMF characterized by periods where disease symptoms are severe (crisis) followed by periods where the disease remits and all blood measures revert to normal (remission). Here, we will elucidate the mechanisms precipitating BMF during crisis periods and mechanisms that mediate the complete recovery of blood formation during remission. Our studies will ultimately provide new therapeutic targets for both inherited and acquired BMF syndromes.
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