The molecular pathophysiology of the congenital dyserythropoietic anemias

University Of Michigan At Ann Arbor, Ann Arbor MI

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Abstract

Project Summary Anemia due to defects in erythropoiesis (red blood cell production) is a major source of mortality and morbidity worldwide. The Congenital Dyserythropoietic Anemias (CDAs) are a group of disorders of terminal erythroid maturation defects characterized by ineffective erythropoiesis and distinctive bone marrow findings. Multiple CDA subtypes exist; CDAII is the most common variant, followed by CDAI. Despite the identification of the genetic defects underlying CDAI and CDAII, the pathophysiology of these disorders remains largely unknown. CDAII is an autosomal recessive disease resulting from mutations in SEC23B, encoding a core component of coat protein complex-2 (COPII) vesicles, which transport secretory proteins from the Endoplasmic reticulum to the Golgi apparatus. We generated a SEC23B deficient human erythroid HUDEP2 cell line. SEC23B deficient HUDEP2 cells survive and expand normally when maintained as proerythroblasts in expansion media; however, upon culturing the cells in differentiation media, these cells exhibit reduced viability and impaired differentiation, as seen in human CDAII. Using this cell line, in preliminary results, we performed a genome- scale CRISPR knock-out screen to identify genes that, when deleted, rescue the CDAII differentiation defect. LRF and miR-451 were 2 of the top hits of the CRISPR screen, which validated in additional preliminary experiments. In aim 1 of this proposal, we propose to define the mechanism by which deletion of LRF and miR- 451 rescue the CDAII erythroid defect. Furthermore, we recently showed through studies in mice and human cells that SEC23B overlaps in function with its closely related paralog, SEC23A. We generated a reporter human erythroid HUDEP2 cell line that expresses eGFP-tagged SEC23A from the endogenous SEC23A locus. Using CRISPR activation (CRISPRa), we found that increasing the SEC23A level by as little as 30% is sufficient to ameliorate the CDAII defect. In preliminary findings, we tested ~5,000 compounds from the drug repurposing library for their ability to increase SEC23A. The top hits form this screen included RN1 (LSD1 inhibitor) and decitabine (DNMT1 inhibitor), both of which showed in validation experiments i) >10-fold increased SEC23A expression in erythroid cells differentiated from CD34 hematopoietic stem cells in vitro, and ii) absence of erythroid differentiation defects at doses sufficient to therapeutically increase SEC23A. In aim 2 of this proposal, we propose to define the roles of RN1 and decitabine in rescuing the CDAII erythroid defect. Finally, we utilized our newly established inducible Gata1-Cre recombinase allele to delete Cdan1 in the adult erythroid compartment, which allowed us to generate the most accurate CDAI model in adult mice, to date. Using this CDAI murine model, we propose several experiments in aim 3 to study the role of CODANIN1 in erythropoiesis. These studies have important implications for understanding the pathophysiology of the CDAs and may lay the foundation for the development of novel therapies for these disorders.