1011 – INVESTIGATION OF HUMAN BONE MARROW PROGENITORS ELUCIDATES CELLULAR AND MOLECULAR MECHANISMS OF ERYTHROID FAILURE IN DIAMOND-BLACKFAN ANEMIA

Abstract

Ribosome dysfunction underlies the pathogenesis of many cancers and heritable ribosomopathies. Diamond-Blackfan anemia (DBA) is a rare bone marrow failure syndrome caused by monoallelic loss-of-function mutations or deletions in small (RPS) or large (RPL) ribosomal protein genes. The molecular mechanisms that underpin differences in clinical phenotypes are yet to be elucidated and therapeutic options are limited. By combining flow-cytometry, cell-sorting and single-cell clonogenic assays of human bone marrow from healthy donors, we identified the immunophenotypic markers that define early and late erythroid progenitors; the cells that give rise to burst- and colony-forming unit-erythroid (BFU-E and CFU-E) colonies respectively. We then applied these definitions to bone marrow derived from children with DBA and elucidated two distinct cellular trajectories segregating with RP genotype: almost complete loss of erythroid lineage specification in RPS-DBA versus relative preservation of qualitatively abnormal erythroid progenitors in RPL-DBA. Single-cell studies of DBA hematopoietic stem and progenitor cells showed that this difference is underpinned by erythroid differentiation arrest and reduced GATA1 activity in RPS-DBA versus preserved GATA1 expression in RPL-DBA. We identified a pro-inflammatory milieu in DBA bone marrow accompanied by P53 activation. The compensatory stress erythropoiesis detected was deficient in its hallmark endogenous glucocorticoid-regulated transcriptional program, leading to disordered differentiation in RPL-DBA. Finally, analysis of patients in the UK DBA registry (n=106) showed that RPL genotype is an independent predictor of older age at presentation with anemia and more frequent and durable responses to corticosteroids. In summary, we present unbiased charting at single-cell resolution of erythropoiesis in DBA bone marrow. Our data categorize developmental trajectories and in turn define genotype-phenotype correlations to facilitate precision-based therapeutic approaches in a rare heritable disease. Ribosome dysfunction underlies the pathogenesis of many cancers and heritable ribosomopathies. Diamond-Blackfan anemia (DBA) is a rare bone marrow failure syndrome caused by monoallelic loss-of-function mutations or deletions in small (RPS) or large (RPL) ribosomal protein genes. The molecular mechanisms that underpin differences in clinical phenotypes are yet to be elucidated and therapeutic options are limited. By combining flow-cytometry, cell-sorting and single-cell clonogenic assays of human bone marrow from healthy donors, we identified the immunophenotypic markers that define early and late erythroid progenitors; the cells that give rise to burst- and colony-forming unit-erythroid (BFU-E and CFU-E) colonies respectively. We then applied these definitions to bone marrow derived from children with DBA and elucidated two distinct cellular trajectories segregating with RP genotype: almost complete loss of erythroid lineage specification in RPS-DBA versus relative preservation of qualitatively abnormal erythroid progenitors in RPL-DBA. Single-cell studies of DBA hematopoietic stem and progenitor cells showed that this difference is underpinned by erythroid differentiation arrest and reduced GATA1 activity in RPS-DBA versus preserved GATA1 expression in RPL-DBA. We identified a pro-inflammatory milieu in DBA bone marrow accompanied by P53 activation. The compensatory stress erythropoiesis detected was deficient in its hallmark endogenous glucocorticoid-regulated transcriptional program, leading to disordered differentiation in RPL-DBA. Finally, analysis of patients in the UK DBA registry (n=106) showed that RPL genotype is an independent predictor of older age at presentation with anemia and more frequent and durable responses to corticosteroids. In summary, we present unbiased charting at single-cell resolution of erythropoiesis in DBA bone marrow. Our data categorize developmental trajectories and in turn define genotype-phenotype correlations to facilitate precision-based therapeutic approaches in a rare heritable disease.