The focus of the current project is to unravel the mechanism underlying how MLL fusion proteins are regulating epigenetic modifications on DNA and chromatin in acute myeloid leukemia (AML) cells. It is already established that AML is associated with early aberrations i.e. rearrangements of key epigenetic regulators such as the Mixed Lineage Leukemia (MLL) gene, which encodes a histone methyltransferase that maintains active chromatin marks at histone H3 on lysine 4 (H3K4) at promoters and enhancers.

It is not possible to study the dynamic recruitment of transcription factors and epigenetic factors in a static system such as isolated AML cells from a patient, because the aberrant epigenetic pattern has already been established in the AML cells. Therefore, a dynamic system is required to understand the initiation of the disease. For this reason, we have established a clinically relevant and dynamic human model system to investigate the epigenetic leukemia-initiating event and how the cells progress to AML, with MLL chromosomal translocations. In collaboration with Mark Chao at Stanford University, we have cultured inducible pluripotent stem cell (iPSC) lines from AML patients with MLL-AF9 and MLL-AF10 translocations in our lab (Chao M et al 2017). The AML-derived iPSCs display a normal epigenetic landscape and phenotype. However, when they are induced to differentiate into the myeloid lineage they re-develop AML characteristics and obtain an AML-specific epigenetic pattern. This unique model thus provides excellent potential to study molecular mechanisms during the development of AML. The AML–iPSC model has been set up in our laboratory, including the myeloid differentiation protocol. Our model is one of the few models in the world that mimics the development of AML in humans.

Up to date, we have expanded the AML-iPSC clones and the normal iPSC control and tested their 12-day differentiation into hematopoietic progenitors. We have also collected cells at day 0, day 8, day 10 and day 12 after differentiation. Currently, we are analyzing gene expression by cap analysis of gene expression (CAGE) in the various differentiation states. In CAGE, the 5’ end of mRNA is sequenced which allows for base pair resolution detection and expression quantification of promoter as well as enhancer regions, hence CAGE will allow us to determine the transcriptome profiling and enhancer activity to understand which genes and pathways are involved in the sequential acquisition of the leukemic transcriptome upon development of AML. Moreover, we will reconstruct the transcriptional regulatory network (TRN) that governs AML stem cells by examining the promoters of differentially expressed genes for the presence of transcription factor binding sites, which makes it possible to infer regulatory edges in the regulatory network.

The aim of this master project is to implement CRISPR-Cas9 technology to genetically and epigenetically manipulate AML-iPSCs to validate the putative role for AML development of several transcription factors, previously found by our ongoing CAGE analysis.

Transcriptional regulators that potentially contribute to leukemogenesis will be deleted with CRISPR-Cas9 and the phenotype will be analyzed. All CRISPR manipulations will be performed at the iPSC stage in the AML-iPSC model. The impacts of CRISPR manipulations will be analyzed by fluorescence-activated cell sorting (FACS) analysis after the induction of myeloid differentiation. In addition, the impacts of epigenetic and genetic manipulation on self-renewal and proliferation will be determined by cell cycle analysis and colony forming cell assay conducted with IMDM-based methylcellulose medium.

In conclusion, this 5 month-master project will allow the student to learn:

- Culture and differentiation methods for iPSC cells
- CRISPR-Cas9 genome editing technique
- Fluorescence-activated cell sorting (FACS) for several analysis
- Transcriptomic analyses by CAGE and validation by quantitative real-time PCR
- Designing and critically analyzing their own experimental set-up