PROJECT SUMMARY/ABSTRACT
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.