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Meningioma 1 (MN1) is an independent prognostic marker for normal karyotype acute myeloid leukemia (AML), with high expression linked to all-trans retinoic acid resistance and poor survival. MN1 is also a potent and sufficient oncogene in murine leukemia models, strongly dependent on the MEIS1/AbdB-like HOX protein complex to transform common myeloid progenitors, block myeloid differentiation, and promote leukemic stem cell self-renewal. To identify key genes and pathways underlying leukemic activity, we functionally assessed MN1 cell phenotypic heterogeneity, revealing leukemic and non-leukemic subsets. Using gene expression profiling of these subsets combined with previously published comparisons of full-length MN1 and mutants with varying leukemogenic activity, we identified candidate genes critical to leukemia. Functional analysis identified Hlf and Hoxa9 as critical to MN1 in vitro proliferation, self-renewal and impaired myeloid differentiation. Although critical to transformation, Meis1 knockdown had little impact on these properties in vitro. However, we identified Meis2 as critical to MN1-induced leukemia, with essential roles in proliferation, self-renewal, impairment of differentiation and disease progression in vitro and in vivo. Here, we provide evidence of phenotypic and functional hierarchy in MN1-induced leukemic cells, characterise contributions of Hlf, Hoxa9 and Meis1 to in vitro leukemic properties, and reveal Meis2 as a novel player in MN1-induced leukemogenesis.

Tracking the behavior of leukemic samples both in vitro and in vivo plays an increasingly large role in efforts to better understand the leukemogenic processes and the effects of potential new therapies. Such work can be accelerated and made more efficient by methodologies enabling the characterization of leukemia samples in multiplex assays. We recently developed three sets of lentiviral fluorescent genetic barcoding (FGB) vectors that create 26, 14, and 6 unique immunophenotyping-compatible color codes from GFP-, yellow fluorescent protein (YFP)-, and monomeric kusabira orange 2 (mKO2)-derived fluorescent proteins. These vectors allow for labeling and tracking of individual color-coded cell populations in mixed samples by real-time flow cytometry. Using the prototypical Hoxa9/Meis1 murine model of acute myeloid leukemia, we describe the application of the 6xFGB vector system for assessing leukemic cell characteristics in multiplex assays. By transplanting color-coded cell mixes, we investigated the competitive growth behavior of individual color-coded populations, determined leukemia-initiating cell frequencies, and assessed the dose-dependent potential of cells exposed to the histone deacetylase inhibitor Entinostat for bone marrow homing. Thus, FGB provides a useful tool for the multiplex characterization of leukemia samples in a wide variety of applications with a concomitant reduction in workload, processing times, and mouse utilization.

Retroviral integration site analysis and barcoding have been instrumental for multiplex clonal fate mapping, although their use imposes an inherent delay between sample acquisition and data analysis. Monitoring of multiple cell populations in real time would be advantageous, but multiplex assays compatible with flow cytometric tracking of competitive growth behavior are currently limited. We here describe the development and initial validation of three generations of lentiviral fluorescent genetic barcoding (FGB) systems that allow the creation of 26, 14, or 6 unique labels. Color-coded populations could be tracked in multiplex in vitro assays for up to 28 days by flow cytometry using all three vector systems. Those involving lower levels of multiplexing eased color-code generation and the reliability of vector expression and enabled functional in vitro and in vivo studies. In proof-of-principle experiments, FGB vectors facilitated in vitro multiplex screening of microRNA (miRNA)-induced growth advantages, as well as the in vivo recovery of color-coded progeny of murine and human hematopoietic stem cells. This novel series of FGB vectors provides new tools for assessing comparative growth properties in in vitro and in vivo multiplexing experiments, while simultaneously allowing for a reduction in sample numbers by up to 26-fold., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)