Inducible MLL-AF9 Expression Drives an AML Program during Human Pluripotent Stem Cell-Derived Hematopoietic Differentiation

Abstract

A t(9;11)(p22;q23) translocation produces the MLL-AF9 fusion protein, which is found in up to 25% of de novo AML cases in children. Despite major advances, obtaining a comprehensive understanding of context-dependent MLL-AF9-mediated gene programs during early hematopoiesis is challenging. Here, we generated a human inducible pluripotent stem cell (hiPSC) model with a doxycycline dose-dependent MLL-AF9 expression. We exploited MLL-AF9 expression as an oncogenic hit to uncover epigenetic and transcriptomic effects on iPSC-derived hematopoietic development and the transformation into (pre-)leukemic states. In doing so, we observed a disruption in early myelomonocytic development. Accordingly, we identified gene profiles that were consistent with primary MLL-AF9 AML and uncovered high-confidence MLL-AF9-associated core genes that are faithfully represented in primary MLL-AF9 AML, including known and presently unknown factors. Using single-cell RNA-sequencing, we identified an increase of CD34 expressing early hematopoietic progenitor-like cell states as well as granulocyte-monocyte progenitor-like cells upon MLL-AF9 activation. Our system allows for careful chemically controlled and stepwise in vitro hiPSC-derived differentiation under serum-free and feeder-free conditions. For a disease that currently lacks effective precision medicine, our system provides a novel entry-point into exploring potential novel targets for personalized therapeutic strategies.

Understanding blood development and leukemia using sequencing-based technologies and human cell systems

“…Recent advancements in genetically modifying hiPSCs unlocked exciting avenues for disease modeling, drug discovery, and regenerative medicine ( Gähwiler et al, 2021 ; Tu et al, 2021 ; Heuts et al, 2023 ). These advancements provide a platform for studying hematopoietic cells that, in some cases, are challenging to genetically manipulate without triggering an immune response.…”

“…This system allows for nearly limitless cell growth and single-cell clonal expansion ( Singh, 2019 ), thus providing a reliable cell source for studying genetically engineered hematopoietic cells. For instance, by integrated doxycycline dose-dependent oncofusion gene expression, e.g., KMT2A-MLLT3 (MLL-AF9) or RUNX1-RUNX1T1 (AML1-ETO), AML programs can be identified in the context of in vitro iPSC-derived hematopoietic differentiation ( Tijchon et al, 2019 ; Heuts et al, 2023 ). Thus, providing insights into blood development and disease mechanisms.…”