Intrinsic and Extrinsic Factors Contributing to Trisomy 21 Preleukemia

PROJECT SUMMARY The initiating genetic alterations leading to blood malignancy occur as early as during fetal development. This is also true in children with Down syndrome (trisomy 21, T21), who have a significantly increased risk of developing preleukemia and leukemia during early childhood. The vision of our project is to identify and

characterize fundamental mechanisms that contribute towards the initiation of preleukemia in Down syndrome children and to establish early therapeutic interventions. Children with Down syndrome have a 150-fold increased risk of developing myeloid leukemia during childhood compared to children without Down syndrome. In 30% of newborns with Down syndrome, a transient

abnormal myelopoiesis (TAM) occurs, which is characterized by a clonal proliferation of immature megakaryoblasts that carry somatic mutations in the transcription factor GATA1. The preleukemic state resolves spontaneously in most newborns but can lead to liver fibrosis and in some cases liver failure. Recently, we

utilized CRISPR/Cas9 editing in human primary fetal liver derived T21 and disomic hematopoietic stem cells (HSCs) to generate an in vivo model of Down syndrome preleukemia and identified the cellular origin of TAM. However, it remains unclear how an extra copy of chromosome 21 predisposes HSCs to blood malignancy, why

somatic mutations are acquired with high frequency and what factors cooperate with GATA1 to initiate TAM. Because the liver is the primary site of hematopoiesis during fetal development, we hypothesize that a combination of intrinsic and extrinsic factors related to T21 and the fetal liver microenvironment contributes to

the higher susceptibility towards preleukemia development. The overall objective of our project is to define specific mechanisms that explain the underlying predisposition of T21 fetal liver HSCs towards initiating preleukemia. In Aim 1, we are proposing to characterize intrinsic factors in T21 HSCs from fetal liver through mapping the gene expression and mutational burden at

single cell resolution. We will assess transcriptional differences in the development and lineages of T21 HSCs compared to disomic fetal liver. In addition, we will quantify the rate of somatic mutations in each subpopulation. In Aim 2, we plan to characterize extrinsic factors in the T21 fetal liver microenvironment through transcriptional

and spatial profiling of the fetal liver. We will carry out functional studies to assess the interaction of stromal components with T21 HSCs. Finally, in Aim 3, we are proposing to functionally evaluate the roles of candidate pathways in preleukemia initiation using our previously established model and primary TAM patient samples.

Our goal is to identify and characterize therapeutic targets for preleukemia and associated liver fibrosis. The proposed research has the potential to change our basic understanding of why Down syndrome children are susceptible to preleukemia and our results could provide a strong rational for subsequent translation

into the clinic.