Single cell LARRY state-fate analysis of Npm1cA/Flt3-ITD acute myeloid leukemia stem cells

AML leukemia stem cells are principal drivers of leukemia disease. Leveraging novel leukemia stem cell cultures (PLSTCs) we performed state-fate analysis to understand the trajectories of distinct AML LSC clones in vivo and ex vivo using a LARRY expressed barcoding strategy. The results indicate that self-renewing LSCs show clonally-heritable programs that predict in vivo engraftment, fitness and therapy response. Targeting those programs using a CROPseq perturbation and single-cell sequencing approach, we identify a novel mechanism regulating the LSC state. Overall design: We first developed PLSTC cultures of AML cells and compared them with standard Stemspan based cultures (experiment 1). Then, we performed multi-indexed LARRY-EGFP(v3) barcoding of inducible Npm1cA/Flt3-ITD hematopoietic stem cells (multiple donors), followed by oncogene activation by Flpo recombinase (experiments 2-5). These origin-barcoded cells were subsequently barcoded using an additional LARRY-Tsapphire barcoding lenti library at day 20. AML cells were processed at day 20 ex vivo (PLSTC cultures, experiment 2), at day 40 in vivo (d20 split-transplant, experiment 3), at day 40 ex vivo (PLSTC cultures, experiment 4), and at day 70 in vivo with and without standard induction chemotherapy (experiment 5). In all these experiments, cells from multiple wells/recipients/donors are multiplexed using TotalSeq cell-hashing (leveraging feature barcoding). In a final experiment, we performed targeted CROPseq to test potential regulators of LSC state-fates (experiment 6).