3131 – KLF1-E325K MUTANT PROTEIN FOUND IN PATIENTS WITH CONGENITAL DYSERYTHROPOIETIC ANEMIA RESULTS IN GENETIC DISARRAY IN ERYTHROBLASTIC ISLAND MACROPHAGES, WITH NO OBSERVED EFFECT ON FUNCTION

Abstract

While the E325K mutation in the transcription factor KLF1 is known to cause morphological defects and gene dysregulation in red blood cells (RBCs), resulting in the disease congenital dyserythropoietic anemia (CDA) type IV, the effect of the mutation on other cell types has not yet been investigated. RBCs develop in erythroblastic islands (EBIs), structures found within the bone marrow consisting of a central macrophage cell surrounded by developing erythroblasts. The macrophage compartment of the EBI niche has been implicated in several RBC disorders, but it was not known whether it is affected by the KLF1-E325K mutation. This question was addressed in two ways. Firstly, to investigate the effect of the mutation on the expression of KLF1 target genes, bulk RNA-sequencing was performed on wild-type iPSC-derived macrophages and mutant iPSC-macrophages from both a CDA patient-derived iPSC line and a KLF1-E325K inducible iPSC line. Secondly, to investigate any functional effects on RBC enucleation and maturation these iPSC-derived macrophages were used in an in vitro model of the human EBI niche. WT-KLF1 and E325K-KLF1 are known to have differences in their ability to activate target genes in RBCs, and this was also observed here in macrophages. For example, FDCSP, encoding a secreted peptide, is upregulated by WT-KLF1 but not by KLF1-E325K. However, using our model of the EBI, E325K mutant macrophages had no detectable functional defects and supported the maturation and enucleation of RBCs at comparable levels to wild-type macrophages. While the E325K mutation in the transcription factor KLF1 is known to cause morphological defects and gene dysregulation in red blood cells (RBCs), resulting in the disease congenital dyserythropoietic anemia (CDA) type IV, the effect of the mutation on other cell types has not yet been investigated. RBCs develop in erythroblastic islands (EBIs), structures found within the bone marrow consisting of a central macrophage cell surrounded by developing erythroblasts. The macrophage compartment of the EBI niche has been implicated in several RBC disorders, but it was not known whether it is affected by the KLF1-E325K mutation. This question was addressed in two ways. Firstly, to investigate the effect of the mutation on the expression of KLF1 target genes, bulk RNA-sequencing was performed on wild-type iPSC-derived macrophages and mutant iPSC-macrophages from both a CDA patient-derived iPSC line and a KLF1-E325K inducible iPSC line. Secondly, to investigate any functional effects on RBC enucleation and maturation these iPSC-derived macrophages were used in an in vitro model of the human EBI niche. WT-KLF1 and E325K-KLF1 are known to have differences in their ability to activate target genes in RBCs, and this was also observed here in macrophages. For example, FDCSP, encoding a secreted peptide, is upregulated by WT-KLF1 but not by KLF1-E325K. However, using our model of the EBI, E325K mutant macrophages had no detectable functional defects and supported the maturation and enucleation of RBCs at comparable levels to wild-type macrophages.