EXPLORING THE PRE-LEUKEMIC PHASE OF ACUTE PROMYELOCYTIC LEUKEMIA: INSIGHTS FROM STUDIES OF SINGLE CELL ¿OMICS¿

Acute Promyelocytic Leukemia (APL) is a distinct subtype of Acute Myeloid Leukemia (AML) driven by the oncogenic fusion protein PML-RAR, which acts as an aberrant transcription factor and blocks myeloid maturation at the promyelocyte stage. In vivo, the expression of PML-RAR is necessary but not sufficient for full-blown leukemia: knock-in (KI) mouse models of APL undergo a prolonged pre-leukemic phase during which hematopoiesis appears normal. This stage provides a unique opportunity to investigate the earliest molecular and cellular alterations that precede the disease. To dissect early events, a transgenic PML-RAR model was used in combination with high-resolution single-cell technologies. Hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), and a residual and more differentiated lineage-negative Sca-1-c-Kit- (LSK-) subpopulation were profiled via bulk RNA-seq, single-cell RNA-seq (scRNA-Seq), and single-cell ATAC-seq (scATAC-Seq). The integrated analysis revealed that PML-RAR expression maintains largely unaltered the overall transcriptional program of pre-leukemic cells, while driving cell cycle and a selective expansion of specific cell subtypes, correlated with a reduction of chromatin accessibility at several genomic loci. Functional assays corroborated these findings. Transplantation experiments demonstrated that PML-RAR-expressing cells from the three hematopoietic compartments gave rise to transplantable leukemia, albeit with different efficiency. Of note, following lethal irradiation, these cells provide radioprotection and long-term repopulation in recipient mice, indicating that PML-RAR confers an advantage that becomes particularly evident in perturbed conditions. Comparison with published datasets further reinforced our observations. A transcriptional signature directly regulated by PML-RAR expression identified in human NB4 APL cells was detected in our analysis of single-cell data. Notably, the signature was confined to the expanded subpopulations, despite differences in species and experimental context. This supports the idea that expanded pre-leukemic progenitors represent the origin of transformation. Together, these results indicate that PML-RAR is able to initiate leukemogenesis through a dual program of chromatin repression and cell cycle dysregulation, selectively expanding progenitor subsets with leukemogenic potential. By integrating transcriptomic, epigenomic and functional data, this work provides a comprehensive map of the earliest oncogenic events of APL, highlighting candidate cell populations from which leukemia may rise. Also, these findings advance the understanding of pre-leukemic biology and suggest novel therapeutic strategies which may intercept disease at its earliest stages.