Pax5 Heterozygosity Delays BCR Expression in Preb-II Cells and Generates a Preleukemic Population

Abstract

Introduction: In B-cells, PAX5 acts as an indispensable master regulator of proliferation and differentiation. In accordance, germline or somatic deregulation of PAX5 facilitates the development of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In this regard, our group has previously established a link between Pax5 and infection exposure in a Pax5+/- mouse model, recapitulating the human disease and providing an explanation for the unique age peak between 2-6 years in pediatric BCP-ALL (Martin-Lorenzo et al., Cancer Discovery, 2015). Nevertheless, the preleukemic setting mediated by reduced Pax5 levels is still poorly understood. Here, we deeply characterized the preleukemic population in Pax5+/- mice utilizing single-cell RNA Sequencing (scRNA-Seq) and multicolor flow cytometry (FACS) analyses.Methods: Aiming to understand the etiology of childhood BCP-ALL in the setting of reduced Pax5 levels, we used 9-color FACS staining and scRNA/bulk RNA-Seq to pinpoint and elucidate a preleukemic population in Pax5+/- mice, which was further investigated after challenges with external stimuli.Results: A comprehensive flow cytometry staining depicting all B-cell developmental stages within the murine bone marrow (BM) showed a robust enrichment of the pre-BII population (B220 +CD19 +IgM -CD25 +) in healthy Pax5+/- mice compared to their wildtype (WT) littermates (Student's t-test; p=0.003). The increased preB-II population could be detected across all ages (3, 6 and 9 months) and the individual cells displayed a higher mean fluorescence intensity (MFI) of CD25 and IL7-Receptor (Student's t-test p=0.0001 and p=0.036, respectively), with almost complete absence of BP-1 (Student's t-test p=0.0001). Bulk RNA-Seq of sorted preB-II populations (3 WT vs. 3 Pax5+/- mice) revealed massive deregulations in major B-cell receptor (BCR) signaling components including downregulation of Cd79a/b, Lyn, MTor and various ITIMs, with concomitant upregulation of Jak3 in Pax5+/- vs WT preB-II cells, which is in line with somatic Jak-Stat activation in infection-driven Pax5+/- leukemias.Additional in-depth characterization of the enriched preB-II population via scRNA-Seq (3 WT vs. 3 Pax5+/- mice, pool of 10,000 cells hashtag labeled, with 50,000 reads/cell and VDJ-rearrangement analysis) furthermore delineated defined sub-developmental stages within the preB-II compartment. In line with the bulk RNA-Seq data, reduced Pax5 levels led to delayed BCR assembly and an arrest of Pax5+/- preB-II cells shortly before BCR expression within the most mature clusters.Finally, challenging Pax5+/- mice with vesicular stomatitis virus (VSV) confirmed the preleukemic identity of this preB-II population, which transformed to a full blown leukemia after additional loss of the second Pax5 allele.Conclusion: In summary, in mice, reduced Pax5 levels generate a preleukemic preB-II population in the bone marrow, which delays BCR rearrangement and confers susceptibility for malignant transformation through infection exposure to drive BCP-ALL development. These findings are important for understanding the molecular mechanisms to prevent or treat a significant proportion of childhood BCP-ALLs. DisclosuresNo relevant conflicts of interest to declare.