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2768 Pax5 Heterozygosity Affects B-Cell Differentiation and Generates a Deregulated Precursor Population in the Bone Marrow

Program: Oral and Poster Abstracts
Session: 603. Lymphoid Oncogenesis: Basic: Poster II
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Lymphoid Leukemias, ALL, Translational Research, hematopoiesis, Diseases, immunology, Lymphoid Malignancies, Biological Processes, molecular biology
Sunday, December 10, 2023, 6:00 PM-8:00 PM

Franziska Auer, PhD1*, Mina Morcos, PhD2*, Mikko Sipola3*, Sanni Moisio, MSc4*, Anna Viitasalo3*, Aleksandra Pandyra5*, Arndt Borkhardt5, Merja Heinäniemi3* and Julia Hauer2*

1Technical University of Munich, School of Medicine, Department of Pediatrics, Neufahrn Bei Freising, Bavaria, Germany
2Technical University of Munich, School of Medicine, Department of Pediatrics, Munich, Germany
3Institute of Biomedicine, School of Medicine, University of Eastern Finland, Yliopistonranta 1, FI-70211, Kuopio, Finland
4Institute of Biomedicine, School of Medicine, University of Eastern Finland, Kuopio, Finland
5Department of Pediatric Oncology, Hematology and Clinical Immunology, Medical faculty, Heinrich Heine University Düsseldorf, Duesseldorf, Germany

Introduction: PAX5 is a well-known master regulator for B-cell development, commitment and identity. Hence, germline or somatic deregulation of PAX5 facilitates the development of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Previously, our group has described B-cell development abnormalities in three families carrying PAX5 germline variants (Escudero et al., Leukemia, 2022) as well as established a link between Pax5 heterozygosity and infection exposure, thereby recapitulating the incomplete penetrance of the human BCP-ALL (Martin-Lorenzo et al., Cancer Discovery, 2015). Nevertheless, the molecular single-cell characteristics of the susceptible precursor population mediated by reduced Pax5 levels is still poorly understood.

Methods: Here, we deeply characterized the susceptible precursor population in Pax5+/- mice utilizing single-cell RNA Sequencing (scRNA-Seq), multicolor flow cytometry analyses and in-vivo transplantation models.

Results: Our data show, that Pax5 heterozygosity leads to an aberrant pre-BII population (B220+CD19+IgM-CD25+) in the bone marrow (BM) of Pax5+/- mice (p=0.0026, Student’s t-test) (Fig. 1A). This population is stable over time and characterized by higher CD25 and IL-7-Receptor expression (p<0.0001, Student’s t-test).

Furthermore, bulk RNA-Sequencing of the pre-BII population revealed B-cell receptor (BCR) signaling as one of the top downregulated pathways (including CD79a/b, Lyn and CD72), while DNA replication and cell cycle signaling pathways were upregulated in Pax5+/- vs. Wildtype (WT) pre-BII cells. ScRNA-Seq analyses confirmed these results (Fig. 1B) and further showed that pre-BII cells of Pax5+/- mice are skewed toward the less commonly used lambda light chain BCR-rearrangements.

The observed delay in the transition to IgM-positivity was additionally validated in a murine in-vivo transplantation model. Here, after 72 hours pre-BII cells from Pax5+/- mice preferably homed to the BM, while matured transplanted WT cells were predominantly found in the spleen (p=0.0023 and p=0.0140, respectively, Student’s t-test). To depict the full spectrum of malignant transformation based on the described deregulated precursor B-cell population, we additionally performed scRNA-Seq on different stages of Pax5-mediated BCP-ALL evolution. Our data show that in a first step, arrested pre-leukemic cells of Pax5+/- mice lose their B-cell identity and display high Myc levels. Sequentially, full blown BCP-ALL arises in a second step after acquisition of additional oncogenic driver mutations (including Jak1 and Jak3). Interestingly, although the analyzed BCP-ALLs of Pax5+/- mice were all based on the same predisposition, they greatly varied in their transcriptional profile and cell-cycle state, depending on their oncogenic 2nd hit.

Conclusion: In summary, we deeply characterized how reduced Pax5 transcriptional activity in the BM generates a predisposed precursor B-cell environment, which is susceptibility for malignant transformation. These findings are important for understanding the molecular mechanisms to prevent or treat a significant proportion of childhood BCP-ALLs.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH