A "De Novo Leukemogenesis" iPSC Model Charts the Clonal Evolution of Acute Myeloid Leukemia
Authors: Wang Tiansu , Kotini Andriana G. , Papapetrou Eirini P. 
Published: October 2019

Abstract
Acute Myeloid Leukemia (AML) develops through the stepwise acquisition of mutations by hematopoietic stem or progenitor cells in a process of clonal evolution, which drives disease initiation, progression and relapse. In recent years, large scale sequencing of patients with AML yielded nearly complete catalogues of the driver mutations, uncovered their temporal occurrences and classified AML subgroups based on mutational profiles. The next daunting challenge is to understand how these mutations contribute to the development of Myelodysplastic Syndrome (MDS) and AML and how the order of their acquisition impacts disease progression.
To study the individual and cooperative effects of driver mutations in MDS and AML and model clonal evolution, we developed iPSC models by introducing mutations in a stepwise manner through serial CRISPR/Cas9-mediated gene editing. We selected two mutational paths, representing de novo AML and secondary AML from preexisting MDS, respectively: (a) the combination DNMT3AR882H, NPM1c and FLT3ITD, the most common 3-gene co-mutation in AML, associated with poor prognosis; and (b) the combination SRSF2P95L, ASXL1 C-terminus truncation and NRASG12D, which represents the "chromatin-spliceosome" AML genomic classification group. The normal iPSC line N-2.12, previously extensively characterized in terms of pluripotency, genetic integrity and hematopoietic differentiation potential, was used as the parental line. Each gene editing step entailed screening of single cell clones by RFLP analysis, confirmation of correct targeting and presence of an intact untargeted allele by DNA sequencing, a second round of single-cell cloning to ensure clonality and karyotyping to exclude chromosomal abnormalities. At least two clones from each step were subjected to hematopoietic differentiation to test for potential phenotypic discrepancies and one representative clone was selected for the next gene editing step.
For the DNMT3AR882H, FLT3ITD, SRSF2P95L and NRASG12D mutations, CRISPR-mediated homology directed repair (HDR) was used. ASXL1mutant lines were generated by CRISPR/Cas9 targeting of the beginning of exon 12 of ASXL1 and selection of clones with frame-shifting indels. NPM1c was introduced with a dox-inducible lentiviral vector. We thus derived a panel of single, double and triple mutant clones representing each step of the clonal evolution of AML.Single mutant clones (DNMT3AR882H and ASXL1-truncated) had completely normal hematopoietic differentiation potential (similar to that of the parental normal line and other non-isogenic normal lines), which is not surprising given that both these mutations are also found in individuals with clonal hematopoiesis without any hematopoietic defects. The double mutant SRSF2P95L-ASXL1 cells showed moderately impaired early hematopoietic differentiation potential with colony-forming ability reduced to approximately 50% of normal. In contrast, both triple mutant lines (DNMT3AR882H-FLT3ITD-NPM1c and SRSF2P95L-ASXL1-NRASG12D) showed a more severe differentiation block with markedly reduced colony-forming ability and prolonged growth in culture for over 11 weeks, in striking contrast to normal iPSC-derived hematopoietic progenitor cells (HPCs) which completely arrest their proliferation after 4 weeks. Transplantation of SRSF2P95L-ASXL1-NRASG12DHPCs into NSG mice resulted in detectable engraftment after 9 weeks in all transplanted animals.
In conclusion, we successfully employed CRISPR/Cas to introduce driver mutations into iPSCs in a stepwise manner to "de novo" reconstruct the development of AML. These panels of iPSCs capture the distinct mutational steps along the evolution of AML in a clonal state and isogenic conditions. They should enable mechanistic studies into the processes of leukemogenesis and the effects of specific mutations acting at distinct stages of this progression, their cooperation and the order by which they are acquired. They should also prove a powerful tool to investigate the minimal genetic requirements for leukemia development, the potential need for cooperating epigenetic insults and the effects of the cellular context in myeloid transformation.

Disclosures
No relevant conflicts of interest to declare.