A pro B cell population forms the apex of the leukemic hierarchy in Hoxa9/Meis1-dependent AML.

Relapse is a major challenge to therapeutic success in acute myeloid leukemia (AML) and can be partly associated with heterogeneous leukemic stem cell (LSC) properties. In the murine Hoxa9/Meis1-dependent (H9M) AML model, LSC potential lies in three defined immunophenotypes, including Lin-cKit+ progenitor cells (Lin-), Gr1+CD11b+cKit+ myeloid cells, and lymphoid cells (Lym+). Previous reports demonstrated their interconversion and distinct drug sensitivities. In contrast, we here show that H9M AML is hierarchically organized. We, therefore, tracked the developmental potential of LSC phenotypes. This unexpectedly revealed a substantial fraction of Lin- LSCs that failed to regenerate Lym+ LSCs, and that harbored reduced leukemogenic potential. However, Lin- LSCs capable of producing Lym+ LSCs as well as Lym+ LSCs triggered rapid disease development suggestive of their high relapse-driving potential. Transcriptional analyses revealed that B lymphoid master regulators, including Sox4 and Bach2, correlated with Lym+ LSC development and presumably aggressive disease. Lentiviral overexpression of Sox4 and Bach2 induced dedifferentiation of H9M cells towards a lineage-negative state in vitro as the first step of lineage conversion. This work suggests that the potency to initiate a partial B lymphoid primed transcriptional program as present in infant AML correlates with aggressive disease and governs the H9M LSC hierarchy.

Real-Time Characterization of Clonal Fate Decisions in Complex Leukemia Samples by Fluorescent Genetic Barcoding.

Clonal heterogeneity in acute myeloid leukemia (AML) forms the basis for treatment failure and relapse. Attempts to decipher clonal evolution and clonal competition primarily depend on deep sequencing approaches. However, this prevents the experimental confirmation of the identified disease-relevant traits on the same cell material. Here, we describe the development and application of a complex fluorescent genetic barcoding (cFGB) lentiviral vector system for the labeling and subsequent multiplex tracking of up to 48 viable AML clones by flow cytometry. This approach allowed the visualization of longitudinal changes in the in vitro growth behavior of multiplexed color-coded AML clones for up to 137 days. Functional studies of flow cytometry-enriched clones documented their stably inherited increase in competitiveness, despite the absence of growth-promoting mutations in exome sequencing data. Transplantation of aliquots of a color-coded AML cell mix into mice revealed the initial engraftment of similar clones and their subsequent differential distribution in the animals over time. Targeted RNA-sequencing of paired pre-malignant and de novo expanded clones linked gene sets associated with Myc-targets, embryonic stem cells, and RAS signaling to the foundation of clonal expansion. These results demonstrate the potency of cFGB-mediated clonal tracking for the deconvolution of verifiable driver-mechanisms underlying clonal selection in leukemia.

A Multiplex CRISPR-Screen Identifies PLA2G4A as Prognostic Marker and Druggable Target for HOXA9 and MEIS1 Dependent AML.

HOXA9 and MEIS1 are frequently upregulated in acute myeloid leukemia (AML), including those with MLL-rearrangement. Because of their pivotal role in hemostasis, HOXA9 and MEIS1 appear non-druggable. We, thus, interrogated gene expression data of pre-leukemic (overexpressing Hoxa9) and leukemogenic (overexpressing Hoxa9 and Meis1; H9M) murine cell lines to identify cancer vulnerabilities. Through gene expression analysis and gene set enrichment analyses, we compiled a list of 15 candidates for functional validation. Using a novel lentiviral multiplexing approach, we selected and tested highly active sgRNAs to knockout candidate genes by CRISPR/Cas9, and subsequently identified a H9M cell growth dependency on the cytosolic phospholipase A2 (PLA2G4A). Similar results were obtained by shRNA-mediated suppression of Pla2g4a. Remarkably, pharmacologic inhibition of PLA2G4A with arachidonyl trifluoromethyl ketone (AACOCF3) accelerated the loss of H9M cells in bulk cultures. Additionally, AACOCF3 treatment of H9M cells reduced colony numbers and colony sizes in methylcellulose. Moreover, AACOCF3 was highly active in human AML with MLL rearrangement, in which PLA2G4A was significantly higher expressed than in AML patients without MLL rearrangement, and is sufficient as an independent prognostic marker. Our work, thus, identifies PLA2G4A as a prognostic marker and potential therapeutic target for H9M-dependent AML with MLL-rearrangement.