Sequential CRISPR gene editing in human iPSCs charts the clonal evolution of leukemia

Human cancers arise through an evolutionary process whereby cells acquire somatic mutations that drive them to outgrow normal cells and create successive clonal populations. “Bottom-up” human cancer evolution models could help illuminate this process, but their creation has faced significant challenges. Here we combined human induced pluripotent stem cell (iPSC) and CRISPR/Cas9 technologies to develop a model of the clonal evolution of acute myeloid leukemia (AML). Through the sequential introduction of 3 disease-causing mutations (ASXL1 C-terminus truncation, SRSF2 P95L and NRAS G12D ), we obtained single, double and triple mutant iPSC lines that, upon hematopoietic differentiation, exhibit progressive dysplasia with increasing number of mutations, capturing distinct premalignant stages, including clonal hematopoiesis, myelodysplastic syndrome, and culminating in a transplantable leukemia. iPSC-derived clonal hematopoietic stem/progenitor cells recapitulate transcriptional and chromatin accessibility signatures of normal and malignant hematopoiesis found in primary human cells. By mapping dynamic changes in transcriptomes and chromatin landscapes, we characterize transcriptional programs driving specific stage transitions and identify vulnerabilities for early therapeutic targeting. Such synthetic “de novo oncogenesis” models can empower the investigation of multiple facets of the malignant transformation of human cells.