Targeting oncogene-induced senescence in ETV6::RUNX1 pre-leukemic cells.

The t(12;21)(p13;q22) is the most common chromosomal translocation in pediatric Bcell precursor acute lymphoblastic leukemia (BCP-ALL), occurring in 2-5% of healthy newborns. This alteration generates the ETV6::RUNX1 (E::R) fusion gene, encoding an aberrant transcription factor that is insufficient to directly cause leukemia, but establishes a clinically silent pre-leukemic progenitor not yet fully characterized. We previously showed that E::R expression in the murine pro-B BaF3 cells caused the slowdown of cell cycle progression and increased phospho-histone H2AX levels, both features of Oncogene-Induced Senescence (OIS). This study investigates E::R's ability to induce senescence in pro-B and immature hematopoietic cells, revealing new therapeutic targets for pre-leukemic cells. We observed that E::R caused a senescence-like phenotype in BaF3 cells, characterized by altered morphology, increased β-galactosidase activity, elevated reactive oxygen species (ROS) and Senescence-Associated Secretory Phenotype (SASP) factor secretion. It dysregulated genes within the p53 pathway, including senescence-related genes, causing the accumulation of p53 protein and alteration in its post-translational modifications. In E::R positive cells, while p53-mediated cell cycle arrest occurred, apoptosis was impaired, providing a survival advantage under genotoxic stress. Multiple therapeutic approaches targeting these vulnerabilities were tested. Senolytics SSK1 and piperlongumine selectively eliminated E::R+ cells by exploiting elevated β-gal activity and ROS levels, respectively. TM5441 leveraged caspase-3 inhibitor PAI-1 upregulation to induce apoptosis. Furthermore, using Sca1-E::R transgenic mice, we validated E::R-induced OIS in the pre-leukemic Lin-Sca1+ immature population and observed reduced pre-B colony formation after SSK1 treatment. These findings demonstrate E::R's dual role in inducing OIS and conferring apoptosis resistance, highlighting the potential of senescence-targeted therapies to prevent leukemia progression and relapse in E::R carriers.