Abstract 30: In-Depth Characterization of B-Lineage Populations Arising During Cord Blood CD34+ HSPC Differentiation for ETV6::RUNX1 Progenitor Identification

Abstract Introduction ETV6::RUNX1 (E::R) B-cell acute lymphoblastic leukemia (B-ALL) frequently arises from a t(12;21)(p13;q22) translocation occurring during fetal development, leading to the formation of dormant preleukemic clones, which only progress to leukemia after accumulating subsequent secondary mutations. Multiple studies have indicated that this pre-leukemic population lies in the CD34+ hematopoietic stem/progenitor cell (HSPC) compartments, however, the exact identity of this progenitor is unknown [Greaves M., 2018]. Objectives An in vitro model for differentiation of cord blood (CB)-derived CD34+CD45(low) HSPCs towards the B-lineage was established. We aim to (a) immunophenotypically elucidate HSPC and B-progenitor sub-populations arising in our model system, (b) detect variances in differentiation potential between GIPFEL-screened E::R+/- CB-HSPCs [Schafer et. al., 2018] and (c) investigate the biological origin of E::R clones. Methods To recapitulate fetal haematopoiesis in vitro, CD34+CD45(low) HSPCs were isolated from CBs (n=5) using magnetic sorting and co-cultured on a human bone marrow MSC (BM-MSC) line in media supplemented with cytokines (Figure 1a.). Flow cytometry protocols were established to distinguish between the HSPC and B-progenitor populations obtained over 35 days of differentiation (Figure 1b.). Single-cell RNA-sequencing (scRNA-Seq) was performed from the mononuclear cell fraction of E::R+ (n=3) and E:R- (n=3) CBs, and will be used to analyze the impact of the translocation on B-lymphopoiesis in cord blood. Results CB-HSPCs were isolated (CD34+ purity 70-90%) and differentiated towards B-cells (~40 % CD19+ cells on day 35) (Figure 1c.) with a 60-fold expansion of hematopoietic cells within the first two weeks (~3*106 CD45+ cells/well on day 14). We identified the various HSPC and naive B-progenitor populations within our system and timepoints of peak emergence of these populations for cell sorting and subsequent experimentation (Figure 1d.). TdT (DNTT) expression was absent during CB-HSPC differentiation, indicative of a fetal differentiation status. Discussion A model system to differentiate CB-HSPCs towards the B-lineage to enable analysis of the earliest progenitors that arise during B-lymphopoiesis was established. We confirmed that our system recapitulates B-cell differentiation during fetal development and identified timepoints for cell sorting of these populations. ScRNA-Seq data will be analyzed to confirm the presence of these populations and understand the nature and impact of the E::R pre-leukemic compartment.