Ontogeny-specific induction of the KMT2A::AFF1-fusion drives development of a distinct CD24 positive pre-leukemic state

Infant Acute Lymphoblastic Leukemia (ALL) driven by the KMT2A::AFF1 onco-fusion is an aggressive, poor prognosis disease with few co-operative mutations. The fusion originates in utero, yet the embryonic initiating steps of disease development remain poorly understood. Here, we present a novel murine KMT2A::AFF1 model, that provides key insights into KMT2A::AFF1 pre-leukemia, relevant to human disease. The model enables precise oncogene induction, and upon targeting hematopoietic stem and progenitor cells (HSPCs) a selective negative impact on proliferation of hematopoietic stem cells (HSCs) was observed, regardless of developmental state during induction. However, a unique CD24 PreProB subset expanded exclusively within the KMT2A::AFF1 embryonic context. This population was absent when targeting lymphoid progenitors, highlighting the importance of the cell of origin for leukemic development. The CD24 PreProB subset displayed key features of pre-leukemic stem cells, including lineage plasticity and aberrant engraftment ability. In line with their pre-malignant phenotype, single-cell transcriptomics revealed a signature consistent with stemness, and notable, up-regulation of Hmga2, a regulator of self-renewal. The signature was critically transferable to human KMT2A::AFF1 patients. Furthermore, given that CD24 is a potential therapeutic target, our findings uncover a distinct embryonic pre-leukemic state with direct relevance to human disease. A murine KMT2A::AFF1 model was used to investigate the impact of the oncogene duringe embryonic development and compared it to adult state. A Col1a1-tetO human KMT2A::AFF1 fusion gene was placed under the control of a Tet-op promoter (KMT2A::AFF1 ). The fusion was made Cre inducible by crossing with the Rosa26-ZtTA strain in which the tetracycline-trans-activator (tTA) contains a STOP cassette flanked by LoxP sites. The expression of KMT2A::AFF1 was induced in embryonic hematopoietic stem and progenitor cells (HSPCs) using Vav-Cre recombinase. Control (cre-)and KMT2A::AFF1+ (cre+) cells from embryo (embryonic day 18.5) and postnatally induced adult mice were stained with CITE seq antibodies (ADTs and HTOs) . Three populations from each developmental time-point and genotype (cre- and cre+) were purified by FACS; LSK, PreProBs and ProBs.