ABSTRACT
Myeloid leukemia in Down syndrome (ML-DS) clonally evolves from transient abnormal myelopoiesis (TAM), a preleukemic condition in DS newborns. To define mechanisms of leukemic transformation, we combined exome and targeted resequencing of 111 TAM and 141 ML-DS samples with functional analyses. TAM requires trisomy 21 and truncating mutations in GATA1; additional TAM variants are usually not pathogenic. By contrast, in ML-DS, clonal and subclonal variants are functionally required. We identified a recurrent and oncogenic hotspot gain-of-function mutation in myeloid cytokine receptor CSF2RB. By a multiplex CRISPR/Cas9 screen in an in vivo murine TAM model, we tested loss-of-function of 22 recurrently mutated ML-DS genes. Loss of 18 different genes produced leukemias that phenotypically, genetically, and transcriptionally mirrored ML-DS.
Subject(s)
Biomarkers, Tumor/genetics , Cell Transformation, Neoplastic/genetics , Chromosomes, Human, Pair 21 , Cytokine Receptor Common beta Subunit/genetics , Down Syndrome/genetics , GATA1 Transcription Factor/genetics , Leukemia, Myeloid/genetics , Leukemoid Reaction/genetics , Mutation , Animals , Disease Models, Animal , Disease Progression , Down Syndrome/diagnosis , GATA1 Transcription Factor/metabolism , Gene Expression Regulation, Leukemic , Genetic Predisposition to Disease , HEK293 Cells , Humans , Leukemia, Myeloid/diagnosis , Leukemia, Myeloid/pathology , Leukemoid Reaction/diagnosis , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Transgenic , Phenotype , Transcription, GeneticABSTRACT
Xenotransplantation is frequently used to study normal and malignant hematopoiesis of human cells. However, conventional mouse xenotransplantation models lack essential human-specific bone-marrow (BM)-microenvironment-derived survival, proliferation, and self-renewal signals for engraftment of normal and malignant blood cells. As a consequence, many human leukemias and other hematologic disorders do not robustly engraft in these conventional models. Here, we describe a complete workflow for the generation of humanized ossicles with an accessible BM microenvironment that faithfully recapitulates normal BM niche morphology and function. The ossicles, therefore, allow for accelerated and superior engraftment of primary patient-derived acute myeloid leukemia (AML) and other hematologic malignancies such as myelofibrosis (MF) in mice. The humanized ossicles are formed by in situ differentiation of BM-derived mesenchymal stromal cells (MSCs). Human hematopoietic cells can subsequently be transplanted directly into the ossicle marrow space or by intravenous injection. Using this method, a humanized engraftable BM microenvironment can be formed within 6-10 weeks. Engraftment of human hematopoietic cells can be evaluated by flow cytometry 8-16 weeks after transplantation. This protocol describes a robust and reproducible in vivo methodology for the study of normal and malignant human hematopoiesis in a more physiologic setting.