Abstract
ZBTB7A is a transcription factor critical for hematopoietic lineage commitment. By binding to promoter regions via its zinc fingers and recruiting corepressors through its BTB domain, ZBTB7A primarily silences its target genes and orchestrates cell fate across several levels and branches of the hematopoietic hierarchy (reviewed in Lunardi et al., 2013; Maeda, 2016). Notably, ZBTB7A is essential for erythroid differentiation at the stage of proerythroblast to erythroblast (Maeda et al., 2009). What is more, ZBTB7A also controls glycolysis, linking differentiation and metabolism (Redondo Monte et al., 2020). In acute myeloid leukemia (AML), ZBTB7A mutations are frequently associated with the t(8;21) translocation, which underlies the RUNX1::RUNX1T1 fusion (also known as AML1-ETO, AE) and defines a patient subgroup with relatively favorable outcomes. However, relapse remains a significant clinical challenge in elderly and medically unfit patients, highlighting the need for novel therapies. Low ZBTB7A expression correlated with worse survival in normal karyotype AML patients ≥60 years old (Hartmann et al., 2016), adding on the interest to understand ZBTB7A loss mechanisms in leukemia and explore them as therapeutic targets.
To investigate the cooperative role of ZBTB7A loss in AE-driven leukemogenesis, we established a murine model by transducing lineage negative (Lin-) bone marrow (BM) from Rosa26-Cas9-EGFP knock-in C57BL/6J mice with a single construct encoding AE (9a splicing variant) and tdTomato (AE9a), accompanied of either a Zbtb7a-targeting (AE9a+KO #1 - #3) or non-targeting sgRNA (AE9a-sgCTRL). The resulting double-positive (DP) EGFP+/tdTomato+ cells of interest were sorted and used for in vitro colony formation (CFU) assays, or transplanted into sub-lethally irradiated C57BL/6J mice via tail vein injection for studying the development of leukemia in vivo.
In vitro, typical blast colonies were absent. However, AE9a+ZBTB7A KO showed a remarkable accumulation of erythroid progenitors (BFU-E) compared to AE9a+sgCTRL in the secondary CFU colonies. We also observed expansion of Zbtb7a KO cells indicated by the increase of indel frequency over replating, indicating that loss of ZBTB7A confers an advantage to AE9a-expressing cells, yet not sufficient for oncogenic transformation in vitro.
In vivo, strikingly, only mice receiving AE9a+ZBTB7A KO cells developed leukemia (median latency: 142 days), whereas control mice (AE9a alone or AE9a-sgCTRL) remained alive for nearly 200 days. Leukemic mice exhibited severe anemia, presence of blasts in the peripheral blood (PB), bone marrow and spleen infiltration (>20% blasts) and splenomegaly. Organ infiltration (commonly spleen and liver, but also lymph nodes) was confirmed by flow cytometry and histopathology. The blasts displayed basophilic cytoplasm resembling erythropoietic precursors, a cytomorphology closely matching that described in the original publication on AE9a-related leukemia (Yan et al., 2006). Conversely, control mice showed mild pallor but no other clinical symptoms. Nevertheless, 85% were anemic at sacrifice, and rare blast-like cells were detected in PB, suggesting a preleukemic state.
Immunophenotyping of BM DP leukemic cells revealed a predominant Sca-1- c-Kit+ CD43++ population, consistent with megakaryocyte-erythroid progenitor (MEP)-like leukemia-initiating cells (Yan et al., 2006; Wang et al., 2022). Besides, about 50% of the DP cells were also CD71+ TER119–, indicating a proerythroblast-stage differentiation block and in accordance with the cytomorphology of the blasts. When investigating committed progenitors within the Lin– Sca-1– c-Kit+ (LK) compartment, a typical MEP population coexisted with an aberrant CD16/32+ CD34– subset, suggesting skewing toward a granulocyte-monocyte progenitor (GMP)-like profile. Recently, Yan and collaborators (2024) have reported a similar skewing of the MEP population towards a GMP fate in their R26-AE9a transgenic mouse model.
In summary, ZBTB7A loss promoted AE9a leukemogenesis likely by enhancing the block of erythroid differentiation characteristic for this model. Ongoing functional assays and single-cell RNA sequencing will further elucidate the nature of RUNX1::RUNX1T1 + ZBTB7A-deficient leukemia, especially regarding the perturbance of upstream progenitor populations, and shed light on specific metabolic features to be explored as novel therapeutic vulnerabilities associated with ZBTB7A loss.
