Functional Interrogation of Human Acute Myeloid Leukemia Stem Cells - PROJECT SUMMARY Studies over the last several decades have led to a model that human AML is organized as a cellular hierarchy initiated and maintained by self-renewing leukemia stem cells (LSCs). The lack of functional studies has limited the understanding of human AML LSCs and the utility of this stem cell model for translational studies in human AML. Recently, we have established technical approaches to enable such functional LSC studies in a manner not previously possible. By employing CRISPR/Cas9 followed by rAAV6-mediated homology directed repair (HDR), we have successfully conducted genome engineering in primary AML cells that can subsequently engraft in vivo. Additionally, we have established iPSCs from several primary AML samples that recapitulate transplantable AML when differentiated into the blood lineage. These AML-iPSCs can be readily engineered using CRISPR/Cas9/HDR to allow for genomic manipulations including inducible Cre-lox for in vivo leukemia gene modulation. Our objectives are to interrogate important questions in human AML stem cell biology. (1) We will investigate the relationship between specific mutations and leukemia stem cell frequency, disease initiation, and disease maintenance. Specifically, we will correct mutations in primary AML cells and/or AML-iPSCs and determine the impact on these parameters. (2) The number and dynamics of individual AML stem cells has not been evaluated at high resolution in vivo. Here, we will investigate in vivo LSC dynamics using methods for single cell lineage tracing including introduction of sequence barcodes using CRISPR/Cas9/HDR. Additionally, we will use mitochondrial single cell ATAC-seq as an endogenous barcoding approach to examine these dynamics directly in patient samples, as well as in xenografted mice and in response to therapy. (3) The primary translational implication of the leukemia stem cell model is that specific targeting of LSCs, even in the absence of targeting of non-LSCs, will lead to eradication of AML. This critical concept will be experimentally investigated by engineering inducible caspase 9 (iCasp9) into the locus for an LSC-specific gene in primary AML cells. Once established in vivo, iCasp9 will be activated to eliminate the LSCs, and the subsequent effects on the leukemia will be determined. These studies should facilitate a deeper interrogation of the leukemia stem cell model in AML and its translational implications.
