Download Noble-Orcutt Klara Abstract 2016

Single-cell analysis reveals molecular mechanisms of NRAS-mediated leukemia stem
cell self-renewal in a murine model of AML.
Klara Noble-Orcutt1, Rebecca LaRue1,2, Conner Hansen1, Ngoc Ha1, Connor Navis1, Alex
Hillesheim1, Fatma Keklik1, David Largaespada2,3 Zohar Sachs1,2
1. Division of Hematology, Oncology, and Transplantation, Department of Medicine
2. Masonic Cancer Center
3. Department of Pediatrics
University of Minnesota
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., 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., 2014). To study NRASmediated leukemia self-renewal, we use a transgenic mouse model of AML with an MllAF9 fusion and a tetracycline repressible, NRASG12V (Kim et al., 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withdrawn.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. One of
these groups (Group 1) preferentially expresses genes associated with leukemia selfrenewal. On the basis of this 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 CD36 -CD69+ LSCs, consistent
with Group 1 gene expression, harbor nearly all of the colony-forming capacity of this
AML. We also transplanted these sorted LSCs into recipient mice and found that only
the CD36-CD69+ LSCs could recapitulate leukemia in mice confirming that this subset of
LSCs is the leukemia stem cell subset. These experiments define the leukemia selfrenewal gene expression profile at the single-cell level and demonstrate how single cell
RNA sequencing can be used to identify critical biological variations among rare cells.