3186 – SINGLE-CELL PROFILING OF T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA WITH CITESEQ IDENTIFIES PRE-LEUKEMIC STEM CELLS

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy driven by the aberrant expression of oncogenic transcription factors. Despite therapeutic advances, T-ALL remains challenging to treat due to the potential for relapse, often driven by leukemic stem cells (LSCs) responsible for initiation of the disease and its recurrence. Understanding the molecular basis of the formation of LSCs is crucial for advancing treatment and identifying potential new molecular targets. In myeloid malignancies, these cells are known to go through a pre-leukemic stage (PLK), also known as pre-LSC, before becoming functional LSCs. The study of PLK status in human T-ALL is particularly challenging due to the inaccessibility of the thymus without invasive techniques. To address this, we applied CITE-seq to a mouse model overexpressing T-ALL oncogenic drivers at different time points during disease development to profile RNA and surface proteins simultaneously. Combined CITE-seq analysis over time revealed the trajectory from PLK to leukemia in T-ALL. It allowed us to identify a small population of cells with stem cell properties compared to other blasts, building a bridge between healthy T-cells and leukemia. Subsequent analysis with Single-Cell Multi-Task Network Inference (scMTNI), a probabilistic method integrating multiple modalities to infer GRN dynamics across cellular trajectories, revealed altered gene regulatory networks (GRN) in these cells. This enabled the identification of stem cell-like properties and highlighted regulators involved in oncogenic and cell-fate specification processes, hallmarks of pre-leukemic stem cells (pre-LSCs). Additionally, through CITE-seq, we identified a set of markers to sort the potential pre-LSC to test their function in vivo.