Acute myeloid leukemia (AML) is frequently fatal because patients who initially respond to chemotherapy eventually relapse. Most anticancer therapies are designed to inhibit proliferation. Yet, in hematopoietic stem cells, the mechanisms of proliferation are distinct from self-renewal (Li et al., Nature 2013). Consequently, targeting proliferation may explain the failure of traditional chemotherapy to eradicate this disease. NRASG12V is required for self-renewal in a murine AML model (Sachs et al., Blood 2014). To study NRAS-mediated leukemia self-renewal, we use a transgenic mouse model of AML with an Mll-AF9 fusion and a tetracycline repressible, NRASG12V oncogene (Kim et al., Blood 2009). Doxycycline abolishes NRASG12V expression leading to leukemia remission. We hypothesize that NRAS-activated pathways required for self-renewal are limited to a subpopulation of LSCs. To identify the NRASG12V-mediated self-renewing subpopulation, we performed single-cell RNA sequencing on the LSC-enriched cells from our AML model. Hierarchical clustering of LSC single-cell data identified three discrete profiles. Comparing the profiles of NRASG12V-expressing LSCs (“RAS-on”) to doxycycline-treated LSCs (“RAS-Off”) revealed that two of the three LSC-expression profiles are lost when NRASG12V is withdrawn. These data suggest that these two profiles are NRASG12V-dependent consistent with an earlier report that activated NRAS exerts bimodal effects on HSCs (Li et al., Nature 2013). One of these LSC subpopulations preferentially expresses genes associated with leukemia self-renewal. On the basis of this single-cell gene expression data, we identfied cell surface markers (CD36 and CD69) that delineate the two NRASG12V-responsive LSC-subpopulations. We sorted LSCs based on CD36 and CD69 status and found that CD36CD69+ LSCs (the group that expresses self-renewal gene expression profiles) harbor nearly all of the colony-forming capacity of the LSCs, forming an average of 13 colonies versus 0.33 colonies for CD69- LSCs and versus 0.11 colonies for non-LSCs (per 10,000 cells plated, p < 0.00001 for each comparison). Accordingly, in vivo mouse reconstituting experiments show that the CD36-CD69+ LSC is the only LSC subgroup that can reconstitute the leukemia in mice (p < 0.005). These experiments characterize the NRASG12V-mediated self-renewal transcriptional signature. Using mass cytometry to query the activation status of signaling pathways simulteneously with multiple immunophenotypic markers, we show that Ki67Low LSCs (the putative self-renewing LSCs) preferentially express increased levels of β-catenin and Myc. These data implicating AML self-renewal pathways can provide precise molecular targets for treating this deadly disease.

Citation Format: Rebecca S. LaRue, Klara E. Noble-Orcutt, Conner Hansen, Ngoc Ha, David A. Largaespada, Zohar Sachs. Single-cell analysis reveals molecular mechanisms of NRAS-mediated leukemia stem cell self-renewal in a murine model of AML. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3339.